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Incorporated  A.D.  1799 


VOLUME  1 6,  PAGES  247-382  APRIL  1911 

Nutrition   Investigations 

on  the 

Carbohydrates 

of 

Lichens,   Algae,   and   Related 
Substances 

BY 

MARY  DAVIES  SWARTZ 

FROM  THE  LABORATORY  OF  PHYSIOLOGICAL  CHEMISTRY 
SHEFFIELD  SCIENTIFIC  SCHOOL 

YALE  UNIVERSITY 

NEW  HAVEN,  CONNECTICUT,  U.  S.  A. 


YALE  UNIVERSITY  PRESS 

NEW  HAVEN,  CONN. 

1911 


5  w  3^ 


COMPOSED   AND   PRINTED   AT   THE 

WAVERLY  PRESS 

By  The  Williams  &  Wilkins  Company 

Baltimobe,  U.  S.  a. 


CONTENTS. 
I.     INTRODUCTION. 

PAGE 

Lichens,  Algae,  Tree  Bark  and  Certain  Tubers  and  Foodstuffs 253 

II.    HISTORICAL  PART. 

Introduction 259 

Cellulose    262 

(a)  Occurrence  and  Nature 262 

(b)  Occurrence  of  Cytases  (Cellulases) 264 

(1)  In  the  Vegetable  Kingdom 264 

(2)  In  Lower  Animals 266 

(3)  In  Higher  Animals .267 

(c)  Digestion  and  Utilization 268 

(1)  By  Animals 268 

(2)  By   Man 269 

The    Pentosans 272 

(a)  Occurrence  and  Nature 272 

(b)  R61e  in  Plant  Physiology 274 

(c)  Occurrence  of  Pentosanases 275 

(1)  In  the  Vegetable  Kingdom 275 

(2)  In  Lower  Animals 276 

(3)  In  Higher  Animals 276 

(d)  Digestion  and  Utilization 278 

(1)  By  Animals 278 

(2)  By  Man 278 

The  Galactans 282 

(a)  Occurrence  and  Nature 282 

(b)  Occurrence  of  Galactanases 284 

(1)  In  the  Vegetable  Kingdom 284 

(2)  In  the  Animal  Kingdom 285 

(c)  Digestion  and  Utilization  by  Animals  and  Man 285 

The  Mannans 289 

(a)  Occurrence  and  Nature 289 

(b)  Occurrence  of  Mannanases 291 

(1)  In  the  Vegetable  Kingdom 291 

(2)  In  the  Animal  Kingdom 292 

(c)  Digestion  and  Utilization  by  Animals  and  Man 293 

The  Levulans 295 

(a)  Occurrence  and  Nature 295 

(b)  Occurrence  of  Levulanases 296 

(1)  In  the  Vegetable  Kingdom 296 

(2)  In  the  Animal  Kingdom 297 

(c)  Digestion  and  Utilization  by  Animals 297 

249 


250  Contents 

The  Dextrans 300 

(a)  Occurrence  and  Nature 300 

(b)  Occurrence  of  Dextranases 301 

(1)  In  the  Vegetable  Kingdom 301 

(2)  In  the  Animal  Kingdom 302 

(c)  Digestion  and  Utilization  by  Animals  and  Man 302 

III.  EXPERIMENTAL  PART. 
Introduction 306 

Chemical  Investigations. 

General  Methods 307 

Pentosan  Preparations 309 

(a)  Dulse    {Rhodymenia  palmata) 309 

(b)  Hawaiian  Seaweeds 313 

(1)  Limu  Lipoa  (Haliseris  'pardalis) 313 

(2)  Limu  Eleele  {Enteromorpha  intestinalis) 313 

(3)  Limu  Pahapaha  (Ulza  lactxica,  etc) 314 

Galactan  Pkeparations 314 

(a)  Irish  Moss  (Chrondus  crispus) 314 

(b)  Hawaiian     Seaweeds 316 

(1)  Limu  Manauea  {Gracilaria  coronopifolia) 316 

(2)  Limu  Huna  {Hypnea  nidifica) 316 

(3)  Limu  Akiaki  (Ahnfeldtia  concinna) 316 

(4)  Limu  Uaualoli  {Gymnogongrus  vermicularis    Americana, 
etc) 316 

(5)  Limu  Kohu  (Asparagopsis  sanfordiana) 316 

(c)  Slippery  Elm  (Ulmus  fulva) 317 

A  Mannan  Preparation — Salep  {Orchis) 318 

A  Levulan  Preparation — Sinistrin  (from  Scilla  Maritima) 321 

Summary    322 

BACTERIOLOGICAL    INVESTIGATIONS. 

Introduction  323 

Trials  with  pure  cultures  of  aerobes 324 

Trials  with  mixtures  of  aerobes 325 

Trials  with  anaerobes 327 

Discussion  and  summary 328 

PHYSIOLOGICAL   INVESTIGATIONS. 

Introduction  331 

Experiments  with  Enzymes 332 

Parental   Injections ' 332 

(a)  Methods  and  Technique 332 


Contents  251 

(b)  Subcutaneous  and  Intraperitoneal  Injections 335 

(1)  Dulse 335 

(2)  Irish  Moss 336 

(3)  Salep 338 

(4)  Sinistrin 340 

Feeding  Experiments 342 

(a)  Methods  and  Technique 342 

(b)  Digestibility  of  Pentosans 344 

(1)  Dulse 345 

(2)  Limu  Eleele 346 

(3)  Litnu  Pahapaha 347 

(4)  Limu  Lipoa 347 

(c)  Digestability  of  Galactans 348 

(1)  Irish   Moss 349 

(2)  Limu  Manauea 350 

(3)  Limu  Huna 351 

(4)  Limu  Akiaki 351 

(d)  Digestibility  of  Mannan 353 

(1)  Salep 354 

Discussion  and  Summary 356 

IV.  CONCLUSIONS. 

V.  BIBLIOGRAPHY. 

Lichens  and  Algae — Composition  and  uses 365 

Cellulose    366 

Pentosans 369 

Galactans  373 

Mannans 376 

Levulans    379 

Dextrans    381 

This  paper  has  been  prepared  from  the  author's  dissertation  submitted  for 
the  degree  of  doctor  of  philosophy,  Yale  University,  1909. 


Digitized  by  tine  Internet  Arciiive 

in  2010  witii  funding  from 

Open  Knowledge  Commons 


http://www.archive.org/details/nutritioninvestiOOrose 


I.  INTRODUCTION. 

Lichens,  Algae,  Tree  Bark  and  Certain  Tubers  as  Foodstuefs. 

From  the  earliest  times,  the  food  of  man  has  included  lichens  and 
algae,  and  even  the  tender  branches  and  inner  bark  of  certain  trees 
and  shrubs,  such  as  elm,  birch,  pine,  and  the  staff-tree  or  bitter-sweet 
{Celastrus  scandens).  When  the  bark  of  trees  is  so  used,  it  is  freed 
from  cork  and  the  hard  outer  rind;  is  cleaned,  dried,  mixed  with  more 
or  less  meal,  and  made  into  "bark  bread,"  Such  substitutes  for 
bread  are  commonly  resorted  to  only  in  northern  lands  where  there  is 
scarcity  of  cereal  crops,  or  in  other  regions  during  periods  of  famine. 
Johnson  (7)  records  that  elm  bark  is  so  employed  in  some  continental 
countries,  and  Dillingham  (4)  relates  that  certain  tribes  of  North 
American  Indians,  'in  times  of  extreme  dearth,  were  accustomed  to 
keep  body  and  soul  together  by  boiling  and  eating  the  bark  of  the 
staff- tree.'  Poulsson  (17)  states  that  in  Finland  and  northern  Russia, 
sphagnum  mosses  are  similarly  employed;  and  Schneider  (21)  agrees 
with  these  other  writers,  saying  that  in  general  lichens  are  used  as 
articles  of  diet  only  in  cases  of  special  need,  principally  because  all 
lichens  contain  a  bitter  principle,  which  not  only  gives  an  unpleasant 
flavor  and  is  difficult  to  remove,  but  also  exerts  an  irritating  effect 
upon  the  digestive  tract,  causing  inflammation.  Nevertheless,  in  the 
northern  parts  of  the  Scandinavian  Peninsula,  where  cereal  crops  are 
always  scanty  or  uncertain,  great  interest  attaches  to  two  species  of 
lichen  widely  distributed  through  Europe,  and  through  Arctic  and 
Antarctic  regions:  namely,  Ceiraria  islandica  and  Cetraria  nivalis, 
which,  as  Poulsson  (17)  observes,  'have  been  considered  nutritive 
and  easily  digestible  since  olden  times. '  Cetraria  islandica,  whitened 
and  freed  from  its  bitter  principle  by  washing  with  dilute  alkali,  is  a 
rather  appetizing  substance;  it  has  sometimes  been  used  as  a  foodstuff 
by  Polar  navigators,  and  Dr.  Hansteen,  chief  lecturer  in  the  Agriciil- 
tural  school  at  Aas,  Norway,  has  gone  so  far  as  to  prophesy  that  moss 
is  destined  to  become  the  great  popular  food  for  the  masses,  because 
of  its  cheapness  and  nutritive  properties. 

Of  marine  algae,  many  tons  are  gathered  and  eaten  annually  in 
various  parts  of  the  world,   the  largest  quantities  being  consumed 

253 


254  Mary  Davies  Swartz, 

by  the  Japanese,  Chinese,  and  Hawaiians.  These  algae  are  found 
in  great  variety  and  widely  distributed.  In  Japan,  the  general  name 
applied  to  them  is  "Nori,"  which  is  also  given  to  several  prepared 
products.  According  to  H.  M.  Smith  (23),  the  most  important  Japan- 
ese seaweed  preparations  are:  "Kanten,"  or  seaweed  isinglass,  made 
from  various  species  of  Gelidium,  the  principal  one  being  Gelidium 
corneum,  often  adulterated  with  similar  seaweeds;  "Kombu"  made 
from  Kelps,  especially  numerous  species  of  Laminaria,  Arthothamnus, 
and  Alaria;  "Amanori,"  from  species  of  Porphyra;  and  "Wakame," 
from  Undaria  pmnatifida. 

Kanten  is  used  largely  for  food,  in  the  form  of  jellies,  and  as  an  adju- 
vant of  soups  and  sauces.  According  to  H.  M.  Smith  (23),  it  is  also 
employed  in  foreign  countries  'in  jellies,  candies,  pastries,  and  many 
desserts,  in  all  of  which  it  is  superior  to  animal  isinglass.'  It  has 
recently  also  attained  popularity  as  a  therapeutic  agent  in  chronic 
constipation,  being  sold  under  various  trade  names,  either  plain  or 
impregnated  with  laxative  drugs,  as  cascara  or  phenolphthalein.i 
Kombu  enters  into  the  dietary  of  every  Japanese  family,  being  cooked 
with  meat,  soups,  etc.,  and  also  served  as  a  vegetable,  or  made  into  a 
relish  with  Soy-bean  sauce.  Amanori  is  eaten  fresh  or  else  is  chopped 
and  sun-dried  in  thin  sheets,  which  are  toastsd  over  a  fire  before 
eating.  The  crisp  amanori  is  crushed  between  the  hands  and  dropped 
into  sauces  or  soups  to  impart  flavor;  or  broken  into  pieces,  dipped  in 
sauce  and  eaten  alone.  Sheets  of  amanori,  spread  with  boiled  rice 
and  covered  with  strips  of  meat  or  fish,  are  rolled  and  cut  into  trans- 
verse slices,  and  take  the  place  of  the  American  sandwich.  Wakame 
is  eaten  as  a  salad,  or  cooked  like  amanori. 

In  Hawaii,  edible  algae  are  called  "limu. "  Of  these  there  are  over 
seventy  distinct  species  used  for  food,  more  than  forty  being  in  general 
use  (18).  Tons  of  limu  are  gathered  for  eating  in  Hawaii  annually, 
and  large  quantities  are  also  imported  from  the  Orient  and  San  Fran- 
cisco. Some  idea  of  the  extent  of  their  use  may  be  gained  from  the 
following  statement  by  Miss  Reed  (18):  "Ancient  Hawaiians  prob- 
ably seldom  ate  a  meal  without  some  kind  of  limu,  and  even  today  no 
Hawaiian  feast  is  considered  quite  complete  without  several  varieties 
served  as  a  relish  with  meats  or  poi."^  Since,  with  the  exception  of  a 
few  experiments  reported  by  Oshima  (15)  and  Saiki  (20),  there  are  no 


iCf.  Galactans,  p.  283. 

^Poi  is  a  thick  paste  made  from  the  root  of  the  taro  plant,  and  takes  the  place  of 
rice  or  bread  in  the  native  diet. 


Nutrition  Investigations.  255 

data  upon  the  digestibility  of  marine  algae,  an  investigation  of  some 
of  these  Hawaiian  limu  seemed  highly  desirable;  and  through  the 
kindness  of  Miss  Reed,  a  number  have  been  obtained  for  this  purpose. 
Their  occurrence  and  uses  will  therefore  be  described  in  some  detail.^ 

These  limu  are  washed  carefiilly  after  gathering,  salted,  and  usu- 
ally broken,  pounded,  or  chopped  into  small  pieces.  They  may  then 
be  eaten  uncooked,  as  a  relish  with  poi,  meats  or  fish;  boiled  with  meats; 
put  into  soups  for  thickening  or  flavoring;  or  roasted  with  pig  in  a  pit. 
Served  raw  and  crisp,  they  take  much  the  same  place  in  the  diet  as 
our  salads.  Among  the  most  popular  varieties  are  Limu  Eleele  {Enter o- 
morpha  of  various  species),  Limu  Kohu  {Asparagopsis  sanfordiana) 
and  Limu  Lipoa  {Ealiseris  pardalis) .  Next  in  favor  come  Limu  Ma- 
nauea  {Gracilaria  coronopifolia) ,  Limu  Buna  {Hypnea  nidifica)  and 
Limu  Akiaki  {Ahnfeldtia  concinna).  Limu  Pahapaha  (Ulva  fasciata 
and  Ulva  lactuca)  is  widely  distributed  but  not  very  popular.  Limu 
Uaualoli  {Gymnogongrus  vermicularis  americana  and  Gymnogongrus 
disciplinalis)  is  limited  to  certain  islands,  and  hence  not  in  such  gen- 
eral use  and  favor  as  some  of  the  others. 

Limu  eleele  is  a  great  favorite,  forming  a  part  of  every  native 
feast.  It  is  generally  eaten  uncooked,  sometimes  being  dropped  into 
hot  gravy,  broth  or  meat  stews  just  before  serving.  Limu  kohu  is 
always  poimded  in  cleaning  to  free  it  from  bits  of  coral  and  soaked  24 
hours  in  fresh  water  to  remove  the  bitter  iodine  flavor.  It  becomes 
sHghtly  fermented  and  acqmres  a  somewhat  sour  taste.  Limu  lipoa 
is  popular  on  account  of  its  penetrating  spicy  flavor,  and  is  frequently 
used  as  a  condiment,  taking  the  place  of  sage  and  pepper  in  Hawaiian 
foods.  Limu  huna  is  especially  prized  for  boiling  with  squid  or  octo- 
pus, though  Umu  manauea  and  Umu  akiaki  are  often  used  as  substi- 
tutes. These  limus,  as  well  as  limu  kohu,  yield  large  amounts  of 
mucilaginous  extract  on  boiling,  limu  manauea  being  considered  es- 
pecially fine  for  thickening  chicken  broth. 

Many  of  the  seaweeds  used  in  Hawaii  and  Japan  occur  also  along 
the  coasts  of  the  United  States  and  Europe,  and  are  to  some  extent 
used  as  food  in  both  regions.  The  very  species  of  Gelidium  from 
which  the  Japanese  prepare  their  Kanten  grow  in  abundance  on  our 
Pacific  coast.  Irish  moss  {Chondrus  crispus),  the  "Tsunomata"  of 
Japan,  has  long  had  considerable  commercial  value  as  a  foodstuff  in 
Ireland.  In  this  country  it  is  found  from  North  Carolina  to  Maine, 
being  especially  abrmdant  north  of  Cape  Cod.    After  cleansing,  cur- 

^For  fuller  description  see  Reed  (18). 


256  Mary  Davies  Swartz, 

ing,  and  bleaching  it  is  to  some  extent  used  for  making  blanc  mange 
or  a  demulcent  for  coughs.  Through  the  kindness  of  Dr.  C.  F.  Lang- 
worthy,  Nutrition  Expert,  United  States  Department  of  Agriculture, 
I  have  obtained  the  foUowing  interesting  data  concerning  the  use  of 
Irish  moss,  from  the  Journal  of  the  South-Eastern  Agricultural  Col- 
lege, Wye,  Kent  (1):  "Professor  D.  Houston,  of  the  Royal  College  of 
Science,  Dublin,  has  favored  us  with  the  following  notes  on  this  sub- 
ject : 

Chondrus  crispus  (carrageen,  or  Irish  moss)  is  a  seaweed  plentifully  distributed 
along  our  northern,  western  and  southern  coasts.  It  is  gathered  and  sold  to  local 
chemists,  who  retail  it,  in  some  parts  at  all  events,  at  6d.  per  pound.  It  is  used  by- 
many  people  as  an  article  of  food  in  the  west,  and  generally  for  colds,  for  which  pur- 
pose it  is  boiled  in  milk. 

Several  of  my  students  tell  me  that  it  is  used  for  feeding  weak  calves  and  with 
striking  results,  bringing  about  an  alteration  of  condition  within  four  days.  One 
student  tells  me  that  in  one  case  at  his  own  farm  a  batch  of  twelve  calves  took  a 
kind  of  wasting  disease,  and  nine  died;  the  other  three  on  the  verge  of  death  were 
given  this  plant,  and  all  three  recovered.  It  is  prepared  by  putting  one  pound  of 
the  "weed"  in  a  net  bag  and  boihng  in  a  gallon  of  water.  The  water  on  cooling 
sets  to  a  jelly.  The  calves  are  given  one  glass  of  jelly  in  their  milk  each  meal 
and  wonderful  results  are  said  to  be  obtained." 

The  high  proportion  of  mineral  matter  is  noteworthy ;i  but  without 
making  a  fuller  investigation,  it  is  impossible  to  say  precisely  wherein 
lies  the  value  of  this  seaweed. 

Purple  laver  {Porphyra  laciniata),  a  source  of  Japanese  amanori, 
is  found  in  abundance  on  the  rocky  shores  of  America  and  Europe 
generally;  but  it  is  not  used  in  this  country  save  sparingly  by  the  Chi- 
nese, who  usually  import  it  directly  from  China,  and  by  some  of  the 
Indians  of  our  northwest  coast.  In  Ireland  it  is  known  as  'sloak,' 
and  is  boiled  and  served  with  butter,  pepper,  and  vinegar  as  an  ac- 
companiment of  cold  meats,  or  is  served  with  leeks  and  onions. 

Dulse  (Rhodymenia  palmata)  is  found  abundantly  on  rocky  shores 
both  in  this  country  and  in  Ireland.  It  is  very  abundant  in  New 
England,  where  it  is  rough-dried  in  the  sun  and  eaten  as  a  relish.  In 
Philadelphia  it  is  caUed  sea-kale  and  eaten  as  a  vegetable.  In  Scot- 
land it  has  long  been  used  both  in  the  fresh  state  and  dried.  In  the 
Scotch  Highlands,  "a  dish  of  dulse  boiled  in  milk  is,"  it  is  said,  "the 
best  of  all  vegetables."  In  Ireland,  it  is  eaten  with  fish  or  boiled  in 
milk  with  rye  flour.  Purple  dulse  {Iridea  edulis),  which  occurs  on 
the  Pacific  coast,  is  often  eaten  like  Rhodymenia  palmata. 

iCf.  Analysis  of  Chondrus  crispus,  p.  254. 


Nutrition  Investigations.  257 

Besides  such  lichens  and  algae,  and  the  bark  of  trees,  various  tubers 
are  used  as  food  for  man.  In  Japan,  the  tubers  of  Hydrosme  rivieri 
(ConophaUus  Konjaku)  are  extracted  with  lime  water,  and  the  result- 
ing gelatinous  mass  is  cut  into  small  cakes.  These,  cooked  with 
"shoyu"  or  Soy-bean  sauce  form  a  common  article  of  diet.  The 
tubers  of  many  species  of  Orchis  and  Eulophia,  native  to  Turkey,  the 
Caucasus,  Asia  Minor  and  the  greater  part  of  Central  and  Southern 
Europe,  furnish  a  food  material  known  as  Salep.  The  small  ovoid, 
oblong  or  palmate  tubers  are  decorticated,  washed,  heated  till  horny 
and  semi-transparent,  and  finally  dried.  An  abundant  mucilaginous 
extract  is  obtained  by  macerating  the  bulbs  in  water.  Frequently 
the  tubers  are  ground  to  powder,  and  the  powder  used  like  sago  or 
tapioca.  Royal  salep,  said  to  be  used  as  food  in  Afghanistan,  is  pre- 
pared from  Allium  Macleanii.  A  former  instructor  in  the  American 
College  for  Girls,  in  Constantinople,  reports  that  salep  is  a  very  com- 
mon article  of  diet  in  Turkey.  It  is  sold  in  the  markets  in  powdered 
form,  and  is  made  into  a  sort  of  sweetened  gruel  with  milk.  Not  only 
is  it  used  as  a  warm  drink  in  the  household,  much  as  we  use  cocoa  or 
chocolate,  but  it  is  also  sold  in  the  streets  by  venders,  who  either 
stand  in  booths  along  the  way,  or  go  about  carrying  huge  brass  urns 
strapped  to  their  shoulders,  clinking  their  cups  and  calling "  Taze- 
Sahlep!"!  It  is  especially  popular  in  districts  of  the  city  where  peo- 
ple work  late  at  night.  In  the  month  of  Ramazon,  the  time  of  all-day 
fasting,  hot  salep  finds  a  ready  sale  at  night.  It  is  no  uncommon 
thing  to  see  the  workman  standing  with  his  salep  cup  in  hand,  waiting 
for  the  firing  of  the  sunset  cannon. 

In  spite  of  the  fact  that  there  have  been  almost  no  scientific  inves- 
tigations as  to  the  digestibiUty  of  such  mucilaginous  plant  substances 
there  seems  to  be  a  special  virtue  attached  to  mucilages  in  the  popular 
mind.  The  prevailing  impression  is  shown  in  some  of  the  following 
remarkable  statements.  The  United  States  Dispensatory,  1908,  not 
only  says  that  the  mucilaginous  extract  of  slippery  elm  hsirk  {Ulmus 
fulva,  Michaux)  is  nutritious,  but  adds,  "We  are  told  that  it  has  proved 
sufficient  for  the  support  of  life  in  the  absence  of  other  food."  Of 
salep  Smith  (25)  says  in  his  dictionary  of  economic  plants:  "It  con- 
tains a  chemical  substance  called  bassorin,  which  is  said  to  contain 
more  nutritious  matter  than  any  other  vegetable  product,  one  ounce 
per  diem  being  sufficient  to  sustain  a  man"!  The  United  States  Dis- 
pensatory also  assures  us  that  salep  is  "highly  nutritious."     Johnson 

^Fresh  salep. 


258  Mary  Davies  Swartz. 

(7)  particularly  recommends  Iceland  moss  {Cetraria  islandica)  as  a 
diet  for  consumptives,  as  "it  seems  to  be  both  extremely  nutritious 
and  very  easy  of  digestion,  though  of  course,  only  capable  of  use  as  a 
substitute  for  starchy  matters."  In  regard  to  Irish  moss  {Chondrus 
crispus),  he  is  a  little  more  uncertain.  "It  is  much  used  for  invalids, 
especially  in  cases  of  consumption,  but  with  doubtful  advantage  when 
substituted  for  more  nutritious  food."  Schneider  (21)  says  of  Ice- 
land moss:  "Inhabitants  of  Iceland,  Norway,  and  Sweden  mixed  this 
lichen  with  various  cereals  and  mashed  potatoes,  from  which  an  un- 
commonly healthful  bread  was  prepared."  Until  the  matter  has  been 
thoroughly  investigated,  we  must  suspend  our  judgment  as  to  the  ac- 
curacy of  such  statements.  After  a  few  metabolism  experiments, 
Oshima  (15)  far  more  conservatively  remarks  concerning  the  algae  of 
Japan:  "Their  actual  value  doubtless  depends  in  considerable  measure 
upon  the  mineral  salts  they  contain." 

In  view  of  the  scarcity  of  any  scientific  investigations  as  to  the  be- 
havior of  all  these  substances  in  the  body,  further  experiments  upon 
their  nature  and  digestibility  seem  highly  desirable,  since  they  are  not 
only  widely  distributed,  and  already  form  a  considerable  portion  of 
the  diet  of  many  persons;  but  because,  if  they  possess  any  real  nutri- 
tive value,  a  wider  use  of  such  comparatively  cheap  materials  would 
be  an  economic  advantage;  and  because,  under  the  prevailing  notions 
as  to  their  food  value,  they  are  sometimes  relied  upon  as  a  source  of 
nutriment  in  diseases  (as  diabetes)  where  the  character  of  the  diet  is 
particularly  important.  The  present  work  has  been  undertaken  to 
throw  some  Hght  on  this  interesting  subject.  A  survey  of  the  litera- 
ture shows  that  even  the  chemical  nature  of  many  of  these  algae  has 
scarcely  been  investigated;  and  if  this  were  known,  we  should  still  be 
under  the  necessity  of  studying  their  behavior  in  the  animal  body, 
for  it  is  impossible  to  tell  from  chemical  analysis  alone  whether  a 
given  substance  will  or  will  not  prove  digestible,  as  Rubner  has  long 
since  warned  us. 


II.   HISTORICAL   PART. 
Introduction. 

According  to  the  current  practice  of  agricultural  analysts,  the  car- 
bohydrates of  plants  are  reported  as  crude  fiber  and  nitrogen-free 
extract.  Crude  fiber  is  the  term  applied  to  the  resistant  mixture  form- 
ing the  mature  cell  wall,  shown  as  long  ago  as  1864  by  Henneberg  and 
Stohman  (41)  to  have  no  definite  chemical  composition.  It  is  there- 
fore not  identical  with  cellulose,  but  consists  of  a  mixture  of  cellulose 
with  incrusting  substances,  lignin  and  cutin,  the  relative  proportions 
of  which  have  recently  been  exhaustively  studied  by  Konig  (51), 
Fiirstenberg  (39),  and  Murdfield  (63).  Cellulose  is  the  chief  consti- 
tuent; the  other  two  are  usually  present  in  varying  proportions. 

Schulze  (74)  to  whom  much  of  our  knowledge  of  the  composition  of 
the  plant  cell  wall  is  due,  has  classified  the  carbohydrates  of  the  nitro- 
gen-free extract  as  follows : 

I.     Water-soluble  carbohydrates.     To  this  class  belong  the  mono-, 
di-,  and  tri-saccharides,  and  some  soluble  polysaccharides. 
II.     Carbohydrates  insoluble  in  water,  but  yielding  sugar  under 
the  action  of  diastase.     The  chief  member  of  this  group  is 
starch. 
III.     Carbohydrates  insoluble  in  water  and  resistant  to  the  action 
of  diastase,  never  being  changed  by  it  into  sugar.    This 
group  is  called  the  Hemicelluloses. 

The  term  hemicellulose,  as  used  by  recent  writers^  seems  to  be  inter- 
preted to  include  some  polysaccharides  of  the  first  group.  It  is  there- 
fore used  here  as  a  group  name  for  those  carbohydrates  which  are  dis- 
tinguished from  cellulose  by  being  capable  of  hydrolysis  on  boiling 
with  dilute  mineral  acids,  and  from  the  other  polysaccharide  carbohy- 
drates by  not  being  readily  digested  by  diastase.  According  to  the 
kind  of  sugar  yielded  on  hydrolysis,  the  hemiceUuloses  are  designated 
as  Pentosans  or  Hexosans,  the  latter  including  Galactans,  Mannans, 
Dextrans,  Levulans,  etc.     After  a  general  review  of  the  chemical 

^e.g.,  Lohrisch. 

259 


260 


Mary  Davies  Swartz, 


nature  of  lichens  and  algae,  each  of  these  classes  will  be  discussed 
separatel);"  in  detail. 

The  percentage  composition  of  some  common  species  of  algae  is 
shown  in  the  following  table : 


WATER. 

PROTEIN. 

FAT. 

CARBOHYDRATES. 

FOOD   MATERIAL. 

^'fref°'       Crude 
Extract. 

ASH. 

I.*       Cystophj'llum    fusiform, 
dried 

15.74 
18.75 

13.53 
23.08 

13.98 

18.92 

80.00 

80.00 

80.00 
13.40 

11.37 

9.58 

19.35 
7.11 

33.75 

11.61 

1.4 

3.7 

1.8 

13.06 

0.32 

.49 
.46 

1.73 

.87 

1.30 
.31 
0.0 

0.0 

0.0 

2.59 
1.2 

54.84 
51.63        9.79 

46.18 
47.70 

41.22 

37.81 

14.4 

12.5 

14.1 
54.16        2.57 
43.3         5.3 

17.56 

Ecklonia  bicyclis,  dried. . 
Enteromorpha   linza, 

dried  (Limu  eleele).. . 
Laminaria  sp.,  dried..  .  . 
Porphyra    laciniata, 

dried 

9.79 

19.21 
21.24 

9.75 

Ulopteryx  pirmatifida, 
dried 

31.35 

Il.t     Ahnfeldtia    concinna, 

fresh  (Limu  akiaki)..  . 
Ulva  fasciata  and  U. 
lactuca,    fresh    (Limu 
Dahapaha) 

4.2 
3.8 

Gracilaria   coronopifolia, 

fresh  (Limu  manauea) 

IILJ   Chondrus  Crispus,  dried. 

IV.  §    Cetraria   islafidica 

4.1 

14.2211 
2.2 

♦Oshiina  (15). 

t  Reed  (18),  (calculated  on  uniform  water  basis). 
tAnnet  (1). 

§  Schmidt  (24),  first  studied  the  ash  and  reported  a  notable  amount  of  calcium  and  potassium 
phosphates.     He  found  no  nitrogen.     Blondeau  (3)  reported  21.36  per  cent    nitrogen. 
II  Brown  (334). 

Until  1905  the  chemical  nature  of  the  constituents  of  algae  had 
received  little  attention.  Analyses  of  many  species  of  algae  from 
Japan  and  China  were  reported  recently  by  Konig  and  Bettels  (8), 
the  results  of  which  are  given  in  the  following  table  on  page  255. 

According  to  Oshima  and  Tollens  (16)  the  carbohydrates  of  Por- 
phyra laciniata  consist  largely  of  anhydrides  of  J-mannose  and  i-ga- 
lactose.  Miither  and  Tollens  (13)  studying  various  species  of  Fucus 
(F.  vesiculosus,  F.  nododus,  F.  serratus),  Laminaria,  and  Chondrus 
crispus,  found  a  methyl-pentosan  (fucosan),  in  Fucus  and  Laminaria; 
and  glucose,  fructose,  galactose  and  pentose  groups  in  Chondrus 
Krefting  reports  a  reserve  carbohydrate  in  Laminaria  digitata  in  win- 


Nutrition  Investigations. 


261 


>-, 

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>> 

43 

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ooououotjuo  y 

333idS3:Sp3  3 

fcnVjUlHl-'l-i'-i'-i'Hti  SH 


O    O 


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o  o  o 


ty  (D  d)  1)  O 

tn  tn  ^  c/a  c/1 

O  O  O  O  ■  O 

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O  U  CJ  CJ  O 

03  rt  _rt  _rt  ^ 

C^  rt  C^  rt  rf 

o  o  o  o  o 


O 


aj    ^    rt 


jrphyra . . 
arphyra  te 
elidium  R 
elidium  B 
elidium  ca 
aminaria 
ther  Lami 

o 

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3  -2 


CqoO-*iO«3t^COC3g 


262  Mary  Davies  Stvartz, 

ter  only,  which  yields  J-glucose.  The  algae  investigated  are  thus  all 
seen  to  yield  pentoses,  very  frequently  fructose  and  methyl-pentose, 
sometimes  glucose  and  galactose. 

Lichens  are  symbiotic  forms  embracing  algae  and  fungi.  Because 
of  this  symbiotic  nature,  they  exhibit  great  variety  in  composition. 
From  the  investigations  of  Escombe  (6),  Ulander  and  Tollens  (27), 
Karl  Miiller  (11),  Nilson  (14),  Wisselingh  (29)  and  others,i  it  appears 
that  the  cell  walls  are  usually  of  cellulose,  but  occasionally  of  chitin.^ 
Many  species  yield  on  extraction  with  hot  water  a  gelatuiizing  sub- 
stance, which  Berzelius  (2)  in  1808  named  "  Flechtenstarke  "  {lichenin), 
but  which  later  investigators^  have  shown  to  be,  not  a  single  substance, 
but  a  number  of  related  carbohydrates  yielding  dextrose,  such  as 
lichenin  from  Cetraria  and  Ramalina  fraxinea,  and  evernin  from  Ever- 
nia  prunastre,  usnin  from  Usnea  barbata.  Other  species,  on  the  con- 
trary yield  little  dextran,  but  mannan,  galactan,  pentosan  and  methyl- 
pentosan  in  varying  proportions.  The  table  on  page  257  showing  the 
hemi-celluloses  occurring  in  a  number  of  lichens,  has  been  compiled 
from  data  given  by  Karl  Miiller  (11)  and  Ulander  and  Tollens  (27). 

Occurrence  and  Nature  or  Cellulose. 

Cellulose  is  said  to  occur  in  pure  form  in  the  wall  of  the  young  plant 
cell.  With  increasing  age,  modifications  take  place  by  which  the  true 
cellulose  becomes  more  and  more  encrusted  with  Hgnin  and  cutin, 
two  substances  shown  by  Konig  (52),  Fiirstenberg  (39),  and  Murdfield 
(63)  to  be  almost  entirely  indigestible.  According  to  Wielen  (87)  and 
Hofmeister  (43),  even  pure  cellulose  is  not  a  simple  substance,  but  can 
be  separated  into  soluble  and  insoluble  portions.'*  Much  of  our  in- 
formation regarding  the  nature  of  cellulose  is  due  to  the  work  of 
Schulze  and  his  pupils.  Schulze  (75)  has  defined  cellulose  as  that  part 
of  the  cell  wall  giving  the  typical  cellulose  reactions,^  and  yielding 
dextrose  on  hydrolysis  with  concentrated  sulphuric  acid. 

Tor  early  literature  see  Czapek,  Biochemie  der  Pflanzen,  Vol.  I,  pp.  514-516. 

-Chitin  occurs  in  Peltigera  canina  and  Evemia  prunastre. 

^Cf.  MuUer  (11)  and  Ulander  (26). 

^According  to  its  beha\'ior  in  sodium  hydroxide  solutions,  the  quantitative  rela- 
tions depending  upon  the  source  of  the  cellulose  and  the  concentration  of  the  solu- 
tion. 

^InsolubiUty  in  dilute  acids  and  alkahes;  solubility  in  ammoniacal  copper  oxide 
solutions;  and  production  of  a  blue  color  with  iodine  and  sulphuric  acid. 


Nutrition  Investigations. 


263 


CO  en     CO     c/2     c/i     en 

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<U  <U     1)     w     <u     <u 


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o  o  o  9 

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5i  fi  ^  c 


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XI  ^  ^  ^ 

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264  Mary  Davies  Sivartz, 

CYTASES   IN   THE   VEGETABLE    KINGDOM. 

By  the  early  investigators,  Haubner  (40),  Henneberg  and  Stohman 
(41),  Kuhn,Aronstein,and  Schulze  (54), it  was  accepted  without  much 
question  that,  since  cellulose  disappeared  from  the  alimentary  tract  of 
herbivora,  it  is  digested  like  starch,  and  equally  valuable  as  a  nu- 
trient. But  after  Tappeiner  (78), in  1884, showed  that  cellulose  could 
be  decomposed  by  micro-organisms,  and  promulgated  his  theory  that 
this  was  the  only  way  to  account  for  the  disappearance  of  cellulose 
from  the  alimentary  canal  of  ruminants,  the  matter  fell  into  great  dis- 
pute,' and  the  question  is  not  yet  definitely  settled  as  to  how  cellulose 
is  digested  and  what  are  the  products  of  its  digestion.  A  diligent 
search  has  been  made  for  enzymes  capable  of  attacking  it  {cytases), 
but  so  far,  such  cytases  have  been  proved  to  exist  only  in  plants  and 
lower  animals.  Many  of  these  so-called  cytases  act  upon  hemicel- 
lulose  rather  than  true  cellulose,  and  will  be  discussed  in  connection 
with  the  hemicelluloses,  though  it  is  not  always  possible  to  make  a 
sharp  distinction  between  the  two.  A  careful  review  of  the  subject 
of  cytases  in  plant  physiology  up  to  1898,  has  been  made  by  Bieder- 
mann  and  Moritz  (34),  from  which  it  appears  that  the  penetration  of 
wood  by  the  mycelia  of  moulds  is  due  to  such  cytases,  and  that  a 
powerful  cellulose-dissolving  enzyme  has  been  derived  from  Peziza 
sclerotium  by  de  Bary  (37)  and  from  another  botrytis  (presumably  a 
Peziza)  by  Ward  (84),  while  Brown  and  Morris  (36)  have  described 
cytases  existent  in  germinating  grasses  which  dissolve  their  cell  walls. 
That  this  is  anything  more  than  a  diastatic  enzyme  is  denied  by  Rei- 
nitzer  (67) ;  but  Newcombe  (64)  considers  the  assumption  of  the  iden- 
tity of  all  cell-wall  dissolving  enzymes  with  diastase  as  far  from  jus- 
tifiable. Bergmann  (32)  'reports  such  cytases  in  hay  and  straw. 
Scheunert  and  Grimmer  (71),  on  the  contrary,  find  none  in  oats,  corn, 
horse-beans,  lupine  seeds,  buckwheat  or  vetch.  Thus  we  see  that 
even  in  the  case  of  plants,  these  enzymes  need  to  be  isolated  and 
identified  before  we  can  arrive  at  any  satisfactory  conclusions. 

That  cellulose  can  be  dissolved  by  bacteria  has  been  demonstrated 
for  such  forms  as  Amylobacter  butyricus,  Vibrio  regula  and  Clostridium 
polymyxa  (34).  Omelianski  (65)  has  described  two  organisms  which 
ferment  cellulose,  and  Ankersmit  (31)  finding  Omelianski's  bacteria 
on  hay,  has  studied  their  behavior  when  introduced  into  the  alimen- 
tary canal  of  the  cow  on  its  food.     He  finds  that  they  do  not  increase 

^For  a  review  of  this  discussion  cf.  Lohrisch  (56). 


Nutrition  Investigations.  265 

in  number  during  their  passage  through  the  digestive  tract,  and  there- 
fore concludes  that  they  play  a  very  inconsiderable  role  in  the  decom- 
position of  cellulose.  According  to  Van  Iterson  (81),  certain  aerobic 
bacteria,  attacking  cellulose,  form  from  it  products  which  nourish 
other  forms  {spirilla);  certain  anaerobes  are  also  shown  to  attack 
it.  Eberlein  (38),  finding  in  the  first  stomach  of  herbivora  Infusoria 
which  utilize  cellulose  for  food,  suggests  that  these  protozoa,  digested 
farther  along  in  the  ahmentary  tract,  serve  as  means  of  transforma- 
tion of  cellulose  into  products  which  the  animal  can  digest;  but  there 
is  nothing  to  indicate  that  such  forms  occur  in  sufi&cient  numbers 
to  be  worthy  of  much  consideration. 

Since  1906  three  investigators  have  given  the  problem  careful  at- 
tention. Scheunert  (68)  has  concluded  from  experiments  in  vitro  that 
bacteria  play  an  exclusive  role  in  the  solution  of  crude  fiber  in  the  coe- 
cal  contents  of  horses,  swine,  and  rabbits.  He  found  that  filtered 
coecal  fluid  acted  on  cellulose  much  less  than  imfiltered  or  simply 
strained  coecal  contents.  This  is  contrary  to  the  opinion  of  Hof- 
meister  (45)  and  Holdefleiss  (48),  who  attribute  the  phenomenon  to 
the  action  of  enzymes,  and  explain  the  loss  of  power  occasioned  by 
filtering  as  due  to  the  effect  of  exposure  to  the  air  upon  the  enzymes. 
Lohrisch  (57)  has  reported  that  fresh  coecal  fluid  is  efi"ective  in  destroy- 
ing cellulose  while  heated  fluid  is  not.  On  the  other  hand,  implanting 
the  sterilized  fluid  with  coecal  bacteria  and  protozoa  would  not  restore 
its  activity.  Coecal  flmd  kept  at  38°  C.  any  length  of  time  gradually 
lost  its  ceUulose-dissolving  power,  while  that  kept  on  ice  remained 
active,  v.  Hoesslin  and  Lesser  (47)  have  attempted  to  explain  these 
apparent  contradictions,  and  conclude  from  their  own  experiments 
that  anaerobic  bacteria  are  the  most  effective  agents  in  cellulose  de- 
composition in  the  intestine.  Equal  volumes  of  non-sterilized  and 
sterilized  coecal  fluid  of  the  horse,  to  which  weighed  amounts  of  cel- 
lulose had  been  added,  were  suspended  in  sterile  physiological  salt  solu- 
tion under  practically  anaerobic  conditions  and  digested  for  periods  of 
from  9  to  35  days.  The  disappearance  of  cellulose  with  the  non-steril- 
ized coecal  fluid  amounted  to  from  55.7  per  cent  to  71.2  per  cent;  with 
sterflized  fluid,  to  from  6.2  per  cent  to  42.4  per  cent.  It  was  also  found 
that  the  addition  of  1-5  grams  of  dextrose  would  effectively  protect 
the  cellulose  from  digestion  by  the  non-sterflized  fluid,  the  bacteria 
preferring  the  more  easily  attacked  carbohydrate.  The  gases  evolved 
in  these  fermentations  were  characteristic  of  bacterial  action,  being 
chiefly  methane,  carbon  dioxide,  and  hydrogen.  The  retarding  effect 
of  exposure  to  the  air  is  explained  by  the  theory  that  anaerobes  are 


266  Mary  Dames  Swartz, 

the  effective  agents.  So,  also,  the  fact  that  Lohrisch  was  unable  to  get 
cellulose  digestion  in  sterilized  fluid  again  inoculated  with  unsteril- 
ized  fluid  is  attributed  to  the  mediiun's  being  an  unfavorable  one  for 
the  development  of  these  organisms,  inasmuch  as  the  addition  of  pep- 
tones to  similar  preparations  caused  in  several  cases  an  increased  de- 
composition. It  seems  fairly  well  estabhshed,  therefore,  that  the 
action  of  the  coecal  fluid  of  the  horse  is  due  to  enzymes  of  bacterial 
origin. 

CYTASES  IN   LOWER  ANIMALS. 

There  is  no  doubt  that  cytases  occur  in  some  of  the  lower  forms  of 
animal  life.  Biedermann  and  Moritz  (34)  fovmd  a  powerful  cellulase 
in  the  secretion  of  the  Hver  of  the  common  snail  {Helix  pomatia),  and 
their  observation  was  verified  by  E.  Miiller  (61),  also  by  Lohrisch  (57) 
who  reports  two  series  of  experiments  in  which  snails  fed  tender  let- 
tuce leaves  digested  from  40.1  per  cent  to  81.6  per  cent  of  the  cellulose 
present.  On  the  other  hand,  Miiller  (61)  could  not  verify  Knauthe's 
report  of  a  cellulase  in  the  hepato-pancreas  of  the  carp  (50) ;  Pacault 
f  otmd  none  in  the  saUva  of  Helix  pomatia  (66) ;  and  Biedermann  none 
in  the  digestive  juice  of  the  meal  worm  {Tenebrio  molitor)  or  of  the 
cabbage  worm  {Fieris  brassica)  (34) .  Biedermann  also  examined  the 
faeces  of  the  cabbage  worm  microscopically  and  foimd  unaltered  par- 
ticles of  leaves,  from  which  he  concluded  that  much  of  the  plant  food 
eaten  is  excreted  unchanged.  Lohrisch  (56)  has  obtained  similar  re- 
sults with  caterpillars  of  sphinx  moths  {Sphinx  euphorbiae),  not  only 
in  experiments  with  intestinal  juice  in  vitro,  but  also  in  feeding  expe- 
riments in  which  the  cellulose  was  quantitively  excreted. 

SeUiere  (75-76)  has  recently  added  some  interesting  contributions 
to  this  subject,  showing  that  cotton  treated  in  various  ways;  namely, 
that  recovered  after  solution  in  Schweitzer's  reagent,  that  treated 
wdth  concentrated  zinc  chloride,  or  with  25  per  cent  caustic  alkali  hot 
or  cold  imtil  the  fibers  are  swollen,  and  subsequently  washed  with 
1  per  cent  acetic  acid  and  water,  is  attacked  by  Helix  pomatia  much 
more  readily  than  the  untreated  substance.  Subsequent  drying  of 
the  treated  cotton  diminished  its  digestibihty  somewhat,  suggesting 
that  the  physical  condition  of  the  cellulose  is  a  definite  factor  in  its 
utilization.  Selliere  believes  that  only  the  more  tender  portions  of 
plant  cellulose  are  attacked  by  the  digestive  juice  of  this  snail.  It 
would  seem  that  the  previous  treatment  of  ths  cellulose  is  a  factor  to  be 
kept  in  mind  in  the  interpretation  of  the  results  of  feeding  experiments.^ 

^Cf.  the  experiments  on  cellulose  utilization  in  the  dog,  p.  263. 


Nutrition  Investigations.  267 

CYTASES  IN  HIGHER  ANIMALS. 

There  is  at  present  no  proof  of  the  existence  of  cytases  in  any  of  the 
higher  animals.  The  literature  on  the  subject  has  been  exhaustively 
reviewed  by  Bergmann  (32),  and  Lohrisch  (55,  56,  57)  audit  appears 
that  there  is  no  cellulase  in  the  saliva  or  pancreatic  juice  of  swine, 
horses,  cattle,  or  sheep.  The  old  observation  by  MacGillawry^  (cited 
by  Biedermann  and  Moritz(34)  that  a  cytase  can  be  extracted  from 
the  vermiform  appendix  of  the  rabbit  has  been  denied  by  Zuntz  and 
Degtiareff  (88).  Schmulewitsch's-  statements  (also  cited  by  Bieder- 
mann and  Moritz)  are  worthless  because  he  employed  no  antiseptics. 
E.  Miiller  (61)  foimd  no  sugar  formed  from  the  decomposition  of  cel- 
lulose in  the  stomach  of  the  goat,  and  Lusk  (59)  observed  no  increase 
in  sugar  elimination  after  feeding  a  phlorhizinized  dog  20  grams  of 
cauUflower,  or  a  phlorhizinized  goat  10  grams  of  paper.  Lohrisch  (57) 
fed  pure  cellulose  (5-20  grams)  to  a  phlorhizinized  rabbit  and  fovmd 
that  it  had  no  marked  influence  on  the  sugar  output,  and  no  nitrogen- 
sparing  effect.  Scheunert  (70)  has  made  further  investigation  on  the 
action  of  the  saliva  and  salivary  glands  in  sheep,  and  confirms  the 
earlier  experiments  with  the  saliva  of  this  animal.  On  the  other  hand, 
Selhere  (77)  reports  that  the  specially  treated  cellulose  mentioned 
above  is  converted  into  dextrose  by  the  intestinal  secretions  of  the 
guinea  pig  in  some  instances. 

Practically  nothing  is  known  concerning  the  way  in  which  cellulose 
disappears  from  the  aHmentary  tract  of  man.  Schmidt  and  Loh- 
risch (73)  fed  pure  cellulose  to  diabetics  and  observed  a  disappearance 
averaging  77.7  per  cent,  and  no  increase  in  the  elimination  of  sugar. 
They  believe  that  most  of  it  is  absorbed  in  soluble  form  and  not  de- 
stroyed by  fermentation  in  the  intestines.  Lohrisch,  having  fed  cel- 
lulose in  various  diseases  of  the  alimentary  tract,^  calls  attention  to 
the  fact  that  in  constipation,  where  there  is  the  least  bacterial  action, 
the  utilization  of  cellulose  is  highest,  while  in  fermentation  dyspepsia, 
in  which  one  might  expect  a  marked  disappearance,  the  utilization  is 
lowest.  He  therefore  considers  the  digestion  of  cellulose  as  due  at 
least  in  part  to  enzymes. 


^Archiv  Neerland,  Vol.  XI. 

^tJber  das  Verhalten  der  Verdauungssafte  zur  Rohfaser  der  Nahrungsmittel. 
Biilletin  de  rAcademie  Imperial  de  St.  Petersburg,  1879. 
^See  results,  p.  264. 


268  Mary  Davies  Swartz, 

Digestion  and  Utilization  of  Cellulose  by  Animals. 

The  literature  on  the  digestion  of  cellulose  up  to  1909  has  been  so 
exhaustively  reviewed  by  Lohrisch  that  it  is  unnecessary  to  enter  into 
a  detailed  discussion  of  it.  From  tables  (55)  showing  the  results  of 
all  previous  experiments  on  the  utiUzation  of  crude  fiber  in  herbivora, 
carnivora,  and  birds,  it  appears  that  in  the  case  of  herbivora,  especi- 
ally ruminants,  20-28  per  cent  of  the  crude  fiber  ingested  with  food 
disappears  from  the  alimentary  canal;  that  in  case  of  carnivora^  and 
birds^  there  is  no  utilization  whatever.  Lohrisch  (56)  himself  reported 
three  experiments  in  which  dogs  were  fed  pure  cellulose  and  digested 
31.1  per  cent,  37.45  per  cent  and  5.4  per  cent  respectively,  but  Scheu- 
nert  and  L5tsch  (72)  repeating  Lohrisch's  work  with  a  somewhat  dif- 
ferent method  of  determining  cellulose  found  that  the  administration 
of  40  grams  of  prepared  white  cabbage,  containing  7.37  grams  of  pure 
cellulose,  resulted  in  the  recovery  of  the  total  amount  ingested.  Cook- 
ing the  cabbage  in  bouillon  did  not  increase  its  digestibility.  They 
attribute  the  apparent  utilization  in  the  preceding  experiment  to  des- 
truction of  cellulose  by  the  reagents  used  for  its  purification.  Since 
the  publication  of  their  paper,  Lohrisch  has  repeated  his  work  with 
the  dog  (57),  and  reports  complete  recovery  of  the  cellulose  fed.  He 
explains  the  error  in  the  earlier  investigation  as  due  to  the  fact  that 
the  ingested  cellulose  was  twice  subjected  to  purification  (before  feed- 
ing and  in  faeces)  with  consequent  increase  in  percentage  of  loss, 
which  was  not  taken  into  account.  He  points  out  the  inevitable  loss 
of  some  cellulose  by  any  method  at  present  in  use  for  its  determina- 
tion, and  defends  his  own  as  sufficiently  accurate  for  all  practical  pur- 
poses if  conditions  are  carefully  observed.^ 

^The  only  experiments  on  record  are  by  Voit  and  Hoffmann  on  the  dog  and  by 
von  Knieriem  on  the  hen. 

^Experiments  by  Weiske  on  the  goose,  and  by  von  Knieriem  on  the  hen. 

'Lohrisch  used  the  method  of  Simon  and  Lohrisch,  in  which  the  cellulose  is  dis- 
solved by  heating  for  an  hour  on  a  water  bath  with  50  per  cent  potassium  hydroxide, 
then  adding  f  cc.  of  30  per  cent  hydrogen  peroxide,  and  digesting  from  §  to  |  hour 
longer  if  necessary.  The  cellulose  is  then  precipitated  by  adding  to  the  solution 
one  half  its  volume  of  96  per  cent  alcohol  and  6-7  cc.  of  concentrated  acetic  acid; 
filtered  off,  washed  with  water,  dilute  acetic  acid,  alcohol  and  ether,  dried  and 
weighed. 

Scheimert  and  Lotsch  mix  the  substance  to  be  analyzed  with  100  cc.  of  cold  water, 
add  100  grams  of  potassium  hydroxide  and  heat  for  an  hour  on  a  water  bath,  then 
filter  through  a  hard  filter  paper,  wash  the  residue  on  the  paper  with  boiling  water 
till  only  a  trace  of  alkali  remains,  transfer  it  to  a  beaker  and  thence  to  a  weighed 


Nutrition  Investigations.  269 

Cellulose  digestion  in  the  dog  has  been  almost  simultaneously  stud- 
ied by  V.  Hoesslin  (46).  Two  dogs  on  a  meat-fat  diet  to  which  was 
added  daily  2  grams  of  specially  prepared  white  cabbage  (containing 
63.25  per  cent  of  pure  cellulose),  for  five  periods  of  five  days  each, 
excreted  on  the  average  99.7  per  cent  and  94.5  per  cent  respectively. 
This  long  experiment  is  significant  as  showing  no  adaptation  of  the 
digestive  glands  to  the  type  of  food.  By  these  independent  workers 
it  seems  now  well  established  that  the  dog  is  unable  to  utilize  cellulose. 

Hoft'mann  (42)  has  just  published  the  results  of  some  investigations 
on  the  influence  of  cellulose  on  the  nitrogen  balance  and  on  phlo- 
rhizin-diabetes  in  the  rabbit,  from  which  it  appears  that  after  inges- 
tion there  is  no  increase  of  sugar  excretion,  and  no  glycogen  formation, 
yet  he  thinks  that  cellulose  and  hemicelluloses  have  a  favorable  influ- 
ence in  phlorhizin-diabetes.^  It  seems  to  follow  from  this,  that  even 
in  case  of  herbivora  cellulose  is  not  utilized  in  the  manner  customary 
for  starch  and  sugar. 

DIGESTION  AND  UTILIZATION   OF   CELLULOSE   BY  MAN. 

A  similar  tabulation  of  results  of  feeding  experiments  on  man,  shows 
that  cellulose  is  not  so  well  utilized  as  by  herbivora,  but  does  disap- 
pear in  appreciable  amounts.  With  one  exception,  the  cellulose  in 
all  these  experiments  was  administered  as  crude  fiber.  Hofmeister 
(43)  fed  pure  cellulose  and  reported  75.7  per  cent  soluble  cellulose  and 
5.6  per  cent  insoluble  cellulose  digested.  Konig  and  Reinhardt  (53) 
added  to  a  diet  rich  in  protein  and  fat,  but  free  from  cellulose,  in  sev- 
eral experiments,  green  peas  and  ripe  shelled  peas,  red  cabbage,  white 

filter,  on  which  it  is  washed  successively  with  hot  water,  dilute  acetic  acid,  hot  water, 
alcohol  and  ether,  and  finally  weighed. 

Scheunert  and  Lotsch  claim  that  by  Lohrisch's  method  the  cellulose  is  altered  in 
character,  and  as  much  as  40  per  cent  lost  in  the  process;  and  that  subsequent  treat- 
ment of  the  recovered  material  causes  an  even  greater  per  cent  of  loss,  while  by  their 
method  the  loss  in  the  first  case  is  not  over  6.8  per  cent,  and  that  in  the  second  case 
even  less. 

For  the  details  of  this  controversy  over  method  see  the  following :  Simon  and  Loh- 
risch;  Zeitschrift  fiir  physiologische  Chemie,  Vol.  42,  p.  55,  (1904).  Scheunert; 
Berliner  tierarztHche  Wochenschrift,  No.  47,  p.  826,  (1909) .  Scheunert  and  Lotsch; 
Ihid.,  p.  867,  (1909);  also  Biochemische  Zeitschrift,  Vol.  20,  p.  10,  (1909);  and 
Zeitschrift  fiir  physiologische  Chemie,  Vol.  65,  p.  219,  (1910).  Scheunert  and 
Grimmer;  Berliner  tierarztHche  Wochenschrift,  No.  48,  p.  152,  (1910).  Lohrisch; 
Zeitschrift  fiir  physiologische  Chemie,  Vol.  69,  p.  143,  (1910). 

^Unfortunately  the  original  paper  was  not  accessible. 


270 


Mary  Davies  Sivartz, 


beans,  graham  and  soldiers'  bread  and  found  30.27  per  cent  to  76.79 
per  cent  of  the  added  cellulose  digested.  Lohrisch  (55)  finds  that  the 
cellulose  of  a  common  vegetable  diet  disappears  from  the  alimentary 
tract  in  large  amounts,  the  actual  quantity  varying  with  the  age, 
source  and  tenderness  of  the  cellulose.  Thus  he  finds  that  for  normal 
indi\dduals,  of  cellulose  from  lentils,  45  per  cent  is  digestible;  from 
kohlrabi,  79.1  per  cent;  from  white  cabbage,  100  per  cent.  Under 
abnormal  conditions  in  the  digestive  tract,  he  has  obtained  the  fol- 
lowing results: 


CONDITION. 

CELLULOSE  UTILIZATION  IN  PER  CENT. 

Normal 

57.9 

Chronic  Constipation 

81.4 

Fermentation  Dyspepsia 

37.8 

Gastrogenic  Diarrhea 

29.5 

Fatty  Faeces  in  Icterus 

27.8 

Fatty  Faeces  in  Disease  of  Pancreas 

20.9 

According  to  Lohrisch,  two  diabetics  on  a  cellulose-free  diet,  to 
which  white  cabbage  was  added  in  quantities  to  yield  about  6  per 
cent  of  cellulose  per  day,  digested  68.6  per  cent  and  84.5  per  cent 
respectively,  without  increased  output  of  sugar  in  the  urine. 

Since  the  only  way  to  determine  definitely  the  energy  value  to  the 
organism  of  such  amounts  of  cellulose  as  are  absorbed,  is  by  means 
of  respiration  experiments,  Lohrisch  (57)  has  performed  such  an  expe- 
riment on  man,  using  the  Zuntz-Geppert  apparatus.  In  fasting,  the 
respiratory  quotient  averages  about  0.76.  After  ingestion  of  carbo- 
hydrates such  as  starch,  it  rises  gradually  in  two  to  three  hours,  to 
0.9-1.0,  and  when  the  carbohydrate  has  been  consumed,  sinks  again 
to  a  lower  level.  Since  the  respiratory  quotient  for  fat  is  0.7  and 
for  protein  about  0.8,  it  is  possible  to  determine  in  this  way  to  what 
extent  the  carbohydrate  replaces  protein  and  fat  in  metabolism. 
Hence  if  cellulose  is  absorbed  and  oxidized  as  a  carbohydrate,  the  res- 
piratory quotient  should  rise.  If  it  is  decomposed  by  bacteria,  the 
respiratory  quotient  should  not  rise,  since  the  theoretical  respiratory 
quotient  for  fatty  acids,  such  as  butyric  and  acetic,  is,  according  to 
Mimk  (62)  and  Mallevre  (60),  0.6  and  0.5  respectively.  Now  Loh- 
risch, feeding  a  man  moist  cellulose  equivalent  to  73.6  grams  of  dry 
substance,  of  which  25  per  cent  was  digested  (18.5  grams)  obtained  the 
following  results: 


Nutrition  Investigations. 


271 


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272  Mary  Davies  Swartz, 

The  respiratory  quotient  attains  its  highest  value  in  the  fourth 
hour,  instead  of  the  second  or  third,  showing  that  cellulose  is  absorbed 
more  slowly  than  starch.  The  rise  is  too  slight  to  indicate  that  cellu- 
lose exercises  any  considerable  protein-  or  fat-sparing  eSect.  It 
is  unfortunate  that  the  amount  of  cellulose  absorbed  was  so  small. 
It  is  striking  that  the  02-consumption  decreases  at  the  very  time  that 
the  respiratory  quotient  rises,  and  the  COo-production  scarcely  in- 
creases. Lohrisch  interprets  this  as  indicating  that  the  increased  02- 
consumption  required  for  oxidation  of  the  cellulose  is  compensated  by 
a  sparing  of  protein  and  fat.  The  difierences  seem  too  small  to  draw 
any  satisfactory  conclusions  as  to  the  energy  value  of  cellulose.  The 
low  respiratory  quotient  in  the  later  hours  of  the  experiment,  together 
vAxh  the  increased  02-consumption,  indicates  the  utilization  of  some 
of  the  cellulose  in  the  form  of  fatty  acids.  We  must  bear  in  mind 
that  no  formation  of  sugar  or  glycogen  from  cellulose,  in  men  or  ani- 
mals, has  been  demonstrated.  Further  investigations  woiild  seem  to 
be  necessary  before  we  can  agree  with  Lohrisch  in  saying,  "  Wir  wissen, 
dass  Cellulose  und  Hemi-cellulosen  vom  Menschen  reichlich  verdaut 
werden,  wir  haben  alien  Grund  anzunehmen,  dass  ihre  Verdauung  nach 
Analogieder  Starke  ahlduf I  .  .  .  Die  resorhirtenM engen  werden  im 
menschlichen  Organismus  vollstdndig  verbrannt.  Dabei  wird  Eiweiss 
und  Fett  von  der  Verbrennung  geschiitzt.^'  In  any  event,  the  quanti- 
ties of  celliilose  which  the  alimentary  tract  of  man  is  capable  of  ab- 
sorbing are,  apparently,  too  small  for  it  to  play  a  role  of  any  impor- 
tance in  the  diet  of  a  normal  individual. 

OCCUREENCE   AND  NATURE   OF  PENTOSANS. 

The  anhydrides  of  the  5-carbon  sugars  are  collectively  designated 
as  pentosans.  These  are  not  reported  to  occur  in  the  animal  kingdom, 
but  the  pentose  sugars  are  found  forming  a  part  of  the  nucleic  acid 
radical  of  the  nucleo-protein  molecule.  In  the  vegetable  kingdom, 
pentosans  are  very  widely  distributed,  as  has  been  shown  by  many 
investigators,  especially  Tollens  and  his  pupils. ^  They  occur  in  all 
kinds  of  plants,  from  the  lowest  to  the  highest,  and  are  limited  to  no 


iToUens,  Landw.  Vers.,  V.  39,  p.  401,  (1891);  Tollens,  Jour.  f.  Landw.,  Vol.  44, 
p.  171  (1896). 

For  an  exhaustive  review  of  the  literature  on  the  occurrence  of  the  pentosans 
see  V.  Lippmann,  Chemie  der  Zuckerarten,  3rd  Edition,  Vol.  I,  pp.  44-60;  116-123; 
and  Czapek,  Biochemie  der  Pflanzen,  Vol.  I,  pp.  537-545  (1905). 


Nutrition  Investigations.  273 

particular  organ  or  tissue,  being  found  abundantly  in  roots,  stems, 
leaves  or  seeds. 

In  regard  to  solubility  in  water,  pentosans  show  all  possible  varia- 
tions. De  Chalmot  (108)  found  them  present  in  the  watery  extract  of 
the  leaves  of  many  plants;  Winterstein  (167)  in  the  somewhat  mucila- 
ginous hot  water  extract  of  the  seeds  of  Tropaeolum  majus;  Schulze  (146) , 
in  both  soluble  and  insoluble  form  in  the  cotyledons  and  endosperms 
of  the  seeds  of  Lupinus  luteus  and  other  legumes,  where  they  are  doubt- 
less stored  as  reserve  material  for  the  growing  plant;  and  in  the  cell 
walls  of  the  mature  plants,  where  in  most  cases  they  approach  true  cel- 
lulose in  character.  It  is  difficult  to  differentiate  these  highly  resis- 
tant pentosans  of  the  cell  wall,  which  are  commonly  included  in  the 
term  crude  fiber,  from  the  hgno-celluloses  and  oxycelluloses  also 
found  there,  which  as  Cross,  Bevan  and  Beadle  (104)  have  shown,i 
are  like  true  pentosans  in  yielding  furfurol  on  distillation  with  dilute 
hydrochloric  acid.  Besides  hemicelluloses  yielding  pentoses  {xylose 
and  arabinose)  exclusively,  occur  many  3delding  also  methyl-pentoses 
(fucose,  rhamnose) .  These  yield  on  distillation  with  dilute  hydrochloric 
acid,  methyl-furfurol,  which  is  precipitated  by  phloroglucin,  and  hence 
included  in  quantitative  estimations  of  pentosans  by  the  method  of 
Tollens  andKrober  (121).  The  distribution  of  methyl-pentosans  has 
been  studied  especially  by  Tollens  and  his  pupils.  Japanese  "Nori" 
{Porphyra  laciniata,  Laminaria,  and  other  seaweeds)  (129),  tragacanth 
and  many  other  gums  (163)  contain  fucosan.  Rhamnose  occurs  also 
widely  distributed  in  the  plant  kingdom,  but  more  frequently  in  the 
form  of  a  glucoside.  Rohmann  (134)  reports  a  rhamnosan  in  Ulva 
lactuca. 

It  is  a  very  common  thing  to  find  pentosans  and  hexosans  occurring 
together.  In  fact,  it  is  absolutely  impossible,  in  treating  of  hemicellu- 
loses, to  draw  any  sharp  dividing  lines,  for  they  are  not  only  intimately 
associated,  but  frequently  chemically  combined.  Schulze  (146)  has 
given  the  name  paragalactan  to  the  carbohydrate  yielding  arabinose 
and  galactose,  which  occurs  in  the  seeds  of  many  legumes.  Winter- 
stein (167)  finds  galacto-xylan  in  the  water  extract  of  Tropaeolum 
majus,  and  numerous  other  examples  of  such  combinations  might  be 
cited. 

A  class  of  substances  to  which  has  been  given  a  distinctive  name 
because  of  their  peculiar  gelatinizing  property,  is  the  Pectins.  As 
Czapek^  remarks,   "It  is  uncertain  whether  they  form  a  definite 


iFor  further  details  see  v.  Lippmann;  Chemie  der  Zuckerarten,  Vol.  I,  pp.  160-169. 
^Die  Pektin-Substanzen;  Czapek,  Biochemie  der  Pflanzen,  Vol.  I,  p.  545. 


274  Mary  Davies  Swartz, 

class  of  cell  wall  substances,  or  whether  they  should  be  classified 
as  'hemicelluloses'  or  'pentosans.' "  In  1868,  Scheibler  (141)  found  a 
sugar  which  he  called  pectinose,  but  which  was  later  shown  to  be  ara- 
binose  (142),  In  1875,  Reichardt  (132)  obtained  a  pectin  body  from 
carrots  and  beets,  which  he  called  'pararabin,'  expressing  the  view 
that  pectins  should  hardly  be  considered  as  a  special  class  of  carbo- 
hydrates. Tromp  de  Haas  and  ToUens  (160)  have  found  from  numer- 
ous analyses,  that  the  pectins  do  not  differ  from  other  carbohydrates 
in  their  relative  proportions  of  hydrogen  and  oxygen  so  much  as  earlier 
workers  supposed,  and  hence  they  may  be  classified  with  other  hemi- 
celMoses  according  to  the  products  of  their  hydrolysis  (pentoses; 
galactose  and  other  hexoses).  Cross  (106)  believes  them  to  be  allied 
to  the  ligno-celluloses.  The  whole  matter  is  still  in  a  state  of  uncer- 
tainty. Herzfeld  (116)  has  shown  that  arabinose  can  be  obtained 
from  most  pectins,  and  consequently  they  have  been  included  among 
the  pentosans,  though  from  the  frequency  with  which  they  yield  ga- 
lactose, they  might  equally  well  be  discussed  with  the  galactans.  Ac- 
cording to  Czapek  while  pectins  occur  frequently  in  phanerogams, 
ferns  and  mosses,  their  presence  in  algae  is  doubtful,  although  it  is 
possible  that  soluble  carbohydrates  of  algae  5delding  arabinose  or  ga- 
lactose are  closely  related  to  the  pectins  of  other  plants. ^ 

Role  of  the  Pentosans  in  Plant  Physiology. 

Comparatively  little  is  known  of  the  role  of  pentosans  in  plant  phys- 
iology. De  Chalmot's  (108)  observation  that  they  decrease  in  quan- 
tity in  seeds  —  peas  and  corn  —  during  germination,  and  reappear 
in  the  stems  and  roots  of  the  growing  plant,  would  seem  to  indicate 
that  they  form  a  part  of  the  reserve  material  in  the  seed;  but  Schone 
and  Tollens  (145),  finding  no  diminution  in  the  amo\mt  of  pentosans 
in  grains  during  germination,  but  rather  a  shght  increase,  declare  that 
they  do  not  belong  to  the  reserve-stuff  of  the  seed;  so  the  question  may 
be  regarded  as  still  unsettled.  Changes  in  the  relative  amounts  of 
pentosan  in  plants  at  different  stages  of  growth,  studied  by  Cross, 
Bevan  and  Smith  (105),  Gotze  and  Pfeiffer  (113),  Calabresi  (98),  and 
others,  show  that  the  increase  of  pentosans  runs  parallel  to  the  forma- 
tion of  the  skeletal  substance ;  and  have  led  to  the  idea  that  they  arise 
through  the  transformation  of  a  part  of  the  cellulose,  and  along  with 
lignin  and  cutin,  take  part  in  wood  formation.     Ravenna  and  Cereser 

^Cf.  also  Bigelow,  Gore,  and  Howard  (92). 


Ntitrition  Investigations.  275 

(131)  find  in  the  case  of  dwarf  beans  that  when  the  food  is  wholly  dex- 
trose administered  to  the  leaves,  pentosans  increase  greatly,  especially 
in  the  light,  and  that  when  the  functioning  of  chlorophyll  is  prevented 
for  long  periods  the  amount  of  pentosans  decreases.  They  conclude 
that  the  simple  sugars  exert  a  preponderating  influence  in  pentosan 
formation,  and  that  these  serve  as  a  reserve  material  when  the  plant 
has  exhausted  its  more  readily  available  food  materials. 

PENTOSANASES  IN   THE  VEGETABLE  KINGDOM. 

Our  knowledge  of  enzymes  inverting  pentosans  is  meager,  and  rather 
indefinite.  The  action  of  such  forms  as  Hymenomycetes  upon  wood 
seems  to  be  of  chemical  nature.  At  any  rate  it  is  evident  (107-146) 
that  they  are  able  to  utilize  xylan.  Bourquelot  and  Herissey  (95)  have 
isolated  an  enzyme  from  malt  diastase  which  produces  reducing  sugar 
from  pectins,  and  call  it  pectinase.  This  is  not  to  be  confused  with 
the  so-called  pectase  which  causes  the  coagulation  of  pectin  bodies. 
Bigelow,  Gore  and  Howard  (92)  also  find  that  the  enzymes  of  Asper- 
gillus partially  hydrolyze  the  pectin  of  gentian  root.  According  to 
Harrison  (114),  Bacillus  (?/grace«  produces  a  cytase  capable  of  dissolv- 
ing the  cell  walls  of  potatoes,  turnips,  cauliflower  and  allied  plants, 
which  acts  particularly  on  the  middle  lamella,  the  supposed  seat  of 
pectin.i  The  latter  is  not  an  inverting  enzyme.  In  Persian  Berries 
(Rhamnus)  (162),  in  Penicillium  glaucum,  and  Botrytis  cinerea  (90), 
an  enzyme  (rhamnase)  has  been  foimd  which  splits  ofl  rhamnose  from 
some  of  its  glucosides  {rhamnetin  and  rhamnazin).  An  early  observa- 
tion of  the  presence  of  rhamnase  in  the  rutin  of  garden  rue  was  made 
by  Borntrager  (94).  That  some  of  the  so-called  cytases  described 
under  cellulose^  may  act  on  pentosans  seems  possible,  but  there  is  no 
direct  evidence  that  such  is  the  case.  On  the  contrary.  Cross  and 
Bevan  (105)  believe  that  pentosans  once  formed  in  the  plant,  remain 
thenceforth  unaltered. 

ToUens  and  Glaubitz  (159)  assert  that  the  pentosans  do  not  undergo 
lactic  or  butyric  acid  fermentation,  and  are  otherwise  unaffected  by 
yeast,  as  has  also  been  shown  by  Lintner  and  Diill  (125).  The  pento- 
sans are  very  resistant  toward  the  action  of  bacteria.  Slowtzoff  (154) 
found  that  a  small  amount  of  pure  xylan  in  a  putrefying  mixture. 


^Cf.  Czapek,  Biochemie  der  Pflanzen. 

-Cf.  Biedermann  and  Moritz  (34),  Brown  (35),  Brown  and  Morris  (36),  Berg- 
mann  (32),  Griiss  (184),  Newcombe  (64). 


276  Mary  Davies  Swariz, 

kept  at  a  temperature  of  40 "^  C,  did  not  entirely  disappear  from  the 
solution  before  the  ninth  or  tenth  day.  Two  widely  distributed  fer- 
menting agents  acting  on  hemicellulose  (Bacillus  aster osporus  Arth. 
Meyer,  and  Bacillus  clostridieforme,  Burri  and  Anker smit),  studied  b}' 
Ankersmit  (89),  are  said  by  him  to  occur  in  insufficient  numbers  to 
make  their  activity  of  any  significance  in  the  alimentary  canal  of  the 
cow. 

PENTOSANASES   IN  LOWER  ANIMALS. 

Extensive  investigations  regarding  the  occurrence  of  pentosan- 
splitting  enzymes  in  lower  animals,  have  been  made  by  Selliere  since 
1905.  The  secretion  of  the  hepato-pancreas  of  the  common  snail 
{Helix  pomatia)  not  only  digests  cellulose  in  vitro, '^  but  also  xylan,  ac- 
cording to  this  writer  (148).  In  feeding  experiments,  analyses  of  the 
food  (oak  wood)  and  excreta  of  these  xylophages  showed  a  higher  per- 
centage of  xylan  in  the  former  than  in  the  latter  (149).  Hence  xylan 
must  have  been  digested.  In  1907,  he  showed  that  pentoses  were 
actually  Uberated  and  absorbed,  by  testing  the  blood  of  these  snails, 
which  gave  the  phloroglucin  reaction  (151).  That  sugar  can  be  found 
in  their  blood  is  denied  by  Couvreur  and  Bellion  (99), but  this  Selliere 
attributes  to  the  fact  that  the  sugar  content  is  much  less  than  in 
higher  animals,  and  hence  has  been  entirely  overlooked. 

Xylanase  also  occurs  in  other  species  of  snail  (150)  such  as  Helix 
aspera  Miill.,  Helix  nemoralis  L.,  Limax  arborum  Bouck.,  Limax 
variegatus  Drap.,  Arion  rufus  L.,  Patella  vulgata  L.,  Littorina  lit'orea  L., 
Littorina  littoralis  L.,  and  in  a  representative  of  the  Coleoptera,  Phy- 
tnatodes  variabilis  L.  The  presence  of  a  xylanase  in  Patella  vulgata 
and  the  Littorinae  is  especially  significant,  as  their  food  consists  in 
pentosan-rich  algae.  Selliere  (150)  and  Pacault  (130)  have  independ- 
ently discovered  a  xylanase  in  the  salivary  glands  of  Helix  pomatia. 
According  to  Rohmann(134),  Aplysia,  which  subsist  largely  upon 
Ulva  lactuca,  do  not,  digest  the  soluble  methyl-pentosan  (rhamnosan) 
present  in  this  alga.  He  finds  this  carbohydrate  present  in  the  glands 
of  the  midgut,  but  regards  it  as  a  food  residue. 

PENTOSANASES  IN  HIGHER  ANIMALS. 

There  have  been  only  a  few  investigations  as  to  the  presence  in 
higher  animals  of  enzymes  hydrolyzing  pentosans.     Slowtzoff  (154) 

^Cf.  Biedermann  and  Moritz  (34). 


Nutrition  Investigations. 


277 


found  that  pure  xylan  was  not  digested  by  saliva,  gastric  or  pancre- 
atic juice,  but  could  be  gradually  hydrolyzed  (in  two  or  three  days)  by 
0.2  per  cent  hydrochloric  acid.  Bergmann  (91)  digested  pure  x>4an 
with  extracts  of  the  intestines  of  many  animals  (hen,  goose,  guinea- 
pig,  sheep,  ox, horse),  and  of  the  vermiform  appendix  of  rabbits,  but  in 
no  case  found  a  xylanase.  These  experiments  were  performed  with 
suitable  antiseptics  and  controls  in  all  cases.  An  old  experiment  by 
Fudakowski  (112),  attributing  an  inverting  action  upon  gum  arabic 
to  pepsin,  and  another  by  Schmulewitsch  (144),  attributing  such  an 
action  upon  crude  fiber  to  pancreatui,must  be  disregarded,  as  no  anti- 
septics whatever  seem  to  have  been  used.  According  to  SeUiere  (152), 
neither  the  pancreatic  juice  of  rabbits,  nor  a  mixture  pancreatic  and  in- 
testinal juices,  will  hydrolyze  xylan.  Negative  results  were  also  ob- 
tained by  him  with  macerated  intestines  of  these  animals.  On  the  other 
hand,  chloroform  extracts  of  the  intestinal  contents  of  rabbits  and 
guinea-pigs  fed  fresh  hay  and  bread,  produced  pentoses  in  a  5  per  cent 
xylan  solution  after  48  hours  digestion  at  37  degrees  C,  while  negative 
results  were  obtained  with  boiled  controls.  This  indicates  that  the 
enzymes  causing  hydrolysis  were  of  bacterial  origin,  a  conclusion  sub- 
stantiated by  later  work  of  the  same  author  (153).  No  xylanase  was 
detected  in  the  excreta  of  carnivora  such  as  the  lion,  panther,  and  wolf. 
From  a  centrifugalized  extract  of  human  faeces  and  soluble  xylan,  di- 
gested imder  aseptic  conditions,  xylose  was  obtained  after  15-20  hours ; 
but  in  meconium  of  calves  and  human  beings  in  which  bacteria  were 
absent  no  xylanase  could  be  found,  although  the  intestinal  glands  were 
functioning.  McCoUum  and  Brannon  (126)  have  shown  that  in  the 
case  of  the  cow  intestinal  bacteria  destroy  pentosans  under  anaerobic 
conditions,  the  degree  of  destruction  var3dng  with  the  kind  of  plant. 
Corn,  wheat  and  oat  feeds  were  incubated  with  fecal  bacteria  of  this 
animal,  and  digestions  continued  14  days  in  atmospheres  both  of  car- 
bon dioxide  and  hydrogen,  with  the  following  average  results : 


MATERIAI.. 

ATMOSPHERE. 

PER  CENT  OF  PENTOSANS  DIS-iPPEARING. 

Corn  Fodder 

CO2 

H 

CO2 

H 

CO2 

H 

51.78 

Corn  Fodder 

Wheat  Straw 

76.13 
28.09 

Wheat  Straw 

37.99 

Oat  Straw 

30.66 

Oat  Straw 

54.00 

From  this  review  it  is  evident  that  the  presence  of  pentosanases  in 
the  higher  animals  has  not  yet  been  demonstrated. 


278  Mary  Danes  Swartz, 

DIGESTION  AND  UTILIZATION   OF  PENTOSANS   BY  ANIMALS. 

In  the  case  of  men  and  animals  subsisting  on  a  mixed  diet,  the  hex- 
oses  and  their  derivatives  so  overbalance  the  pentosans,  under  normal 
conditions,  that  the  utilization  of  the  latter  is  a  question  of  theo- 
retical rather  than  of  practical  importance.  But  in  the  case  of  herbi- 
vora,  limited  to  a  diet  in  which  pentosans  occur  in  considerable 
amoimts,  the  extent  of  pentosan  utilization  becomes  a  question  of 
economic  importance.  It  is  not  surprising  to  find,  therefore,  that 
since  the  development  of  satisfactory  methods  of  quantitative  deter- 
mination, a  considerable  number  of  investigations  have  been  made 
upon  such  utilization  by  animals.  The  results  of  these  experiments 
are  shown  in  tables  on  pages  274  and  275. 

The  results  in  these  experiments  were  obtained  by  analysis  of  food 
and  faeces.  Lmdsey  (123)  Gotze  and  Pfeiffer  (113)  and  ToUens  (157) 
found  no  measurable  amount  of  pentoses  or  pentosans  excreted  in  the 
urine  of  sheep,  but  Neuberg  and  Wohlgemuth  (128)  state  that  pento- 
sans always  occur  in  the  urine  of  rabbits,  only  disappearing  when  the 
vegetable  diet  is  compensated  by  pentose-free  material.  They  report 
that  9  per  cent  of  soluble  araban  (cherry  gum)  fed  to  rabbits  was  ex- 
creted in  the  urine.  Slowtzoff  (154)  found  1.4-4.5  per  cent  of  xylan  in 
the  urine  of  rabbits,  but  no  reducing  sugar.  He  also  found  that  if 
the  animal  were  killed  shortly  after  xylan  feeding,  xylan  could  be  de- 
tected in  blood,  liver  and  muscles.  Hence  xylan  must  have  been  ab- 
sorbed from  the  digestive  tract. 

The  feeding  experiments  show  that  herbivora  digest,  on  the  aver- 
age, 55-60  per  cent  of  the  pentosans  in  their  diet,  but  since  no  animal 
enzymes  hydrolyzing  pentosans  have  been  demonstrated,  and  there 
is-  always  the  possibility  of  bacterial  decomposition  in  the  intestines, 
the  most  conclusive  experiments  as  to  the  actual  nutritive  value  are 
those  of  Kellner  (118)  with  the  respiration  calorimeter.  From  the 
slight  difference  in  loss  of  potential  energy,  when  the  furfurol-yielding 
rye  straw  preparation  was  substituted  for  starch,  he  concludes  that 
furfurol-yielding  substances  participate  in  the  formation  of  fat  in  the 
animal  body. 

DIGESTION  AND  UTILIZATION   OF   PENTOSANS   BY  MAN. 

We  have  seen  that  pentosans  can  be  digested  by  herbivora  to  a 
considerable  extent.  Can  they  be  digested  by  man?  The  only 
feeding  experiments  on  record  are  by  Konig  and  Reinhardt  (120). 


Nutrition  Investigations. 


279 


In  1902,  they  conducted  researches  on  two  men  whose  main  diet  con- 
sisted of  meat  and  butter  or  other  fat,  and  beer;  to  this,  in  the  various 
experiments,  were  added  respectively  (along  with  sugar,  butter,  beef 
extract,  etc.,  used  in  preparing  them)  the  following  substances: 

Experiment  I.  Green  Peas.  Experiment  II.  Ripe  Shelled  Peas. 
Experiment  III.  Red  Cabbage.  Experiment  IV.  Canned  White 
Beans.  Experiment  V.  Soldiers'  Bread.  Experiment  VI.  Graham 
Bread. 

From  analyses  of  food  and  faeces  the  following  results  were  obtained; 


TOTAL  PENTOSANS  IN  GRAMS. 

EXP.  I. 

n. 

m. 

IV. 

V. 

VI. 

In  Food  

15.55 
0.79 

5.08 

7.47 

23.15 
0.59 

2.55 
3.24 

14.01 
0.70 

5.0 

7.75 

12.80 
1.12 

8.75 
14.32 

52.64 
8.66 

16.45 
20.24 

41  26 

In  Faeces  

4.06 

9.84 
12  97 

Percent  not  utilized,  estimating  Pen- 
tosans in  Beer  as  unutilized 

Total  per  cent  not  digested 

Hence  we  see  that  of  the  total  pentosans  in  the  diet  3.24-20.24  per 
cent  were  excreted.  Only  a  little  furfurol-yielding  substance  was 
found  in  the  urine.  From  the  small  percentage  recovered  in  these 
experiments,  Konig  and  Reinhardt  (120)  conclude  that  the  pentosans 
are  to  a  high  degree  utilized  by  man,  but  they  take  no  account  of  pos- 
sible destruction  by  bacteria. ^ 

Since  pentosans  do  disappear  from  the  alimentary  tract  of  men  and 
animals,  it  behooves  us  to  consider  whether,  on  the  assumption  that 
they  are  hydrolyzed  like  starch,  the  pentose  sugars  so  produced  are 
as  well  utilized  as  dextrose.  Konig  and  Reinhardt  (120)  found  some 
furfurol-yielding  substance  in  the  urine,  and  Blumenthal  (93)  observes 
that  after  eating  huckleberries,  cherries  and  prunes,  pentosans  are 
excreted,  but  no  reducing  sugar.  Cominotti  (100)  finds  pentoses  ab- 
sent from  the  urine  of  man  on  a  meat  diet,  but  always  present  on  a 
mixed  diet.  He  agrees  with  Konig  and  Reinhardt  that  the  output  in 
the  urine  is  small  compared  with  the  amount  of  pentosans  in  the  food, 
and  proposes  to  investigate  the  possibility  of  glycogen  formation  from 
pentosans. 

The  behavior  of  pentoses  in  the  body  has  been  exhaustively  reviewed 
by  Neuberg(127).2    It  appears  from  the  work  of  Cremer  (102,  103), 

^  Cramer  (101)  has  shown  (according  to  a  recent  review,  the  original  paper  was 
not  accessible)  that  bacteria  are  essential  to  hemicellulose  transformation. 

^For  a  recent  discussion  of  the  absorption  and  utilization  of  pentoses  see  A.  Mag- 
nus-Levy, Oppenheimer's  Handbuch  der  Biochemie  der  Menschen  und  der  Tiere, 
1909,  Vol.  IV,  pp.  395^07. 


280 


Mary  Davies  Swartz, 


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282  Mary  Davies  Swartz, 

Ebstein  (109),  Frantze  (111),  Neuberg  and  Wohlgemuth  (128),  Sal- 
kowski  (137),  V.  Jacksch  (117),Lmdemann  and  May  (122),Brasch  (96) 
and  others,  that  the  pentoses  and  methyl-pentoses  (rhamnose)  are  ex- 
creted more  readily  than  the  hexoses;  that  they  exert  an  unfavorable 
effect  in  diabetes;  and  that  there  is  no  evidence  of  their  acting  as  gly- 
cogen-formers  in  man.  Consequently,  even  if  further  experiments 
justify  Konig  and  Reinhardt's  conclusions,  the  pentosans  must  appar- 
ently still  play  a  very  small  part  in  the  nutrition  of  man. 

Occurrence  and  Nature  of  Galactans. 

Next  to  the  pentosans,  no  hemicelluloses  seem  to  be  so  widely  dis- 
tributed as  the  galactans;  both  occur  together  in  the  plant  cell,  and 
often  in  a  more  or  less  intimate  chemical  combination.  The  pure 
galactans,  i.e.,  those  yielding  exclusively  galactose  upon  hydrolysis, 
have  been  differentiated  into  several  classes,  chiefly  by  difl'erences  in 
siylubihty  or  specific  rotation,  namely: 

1.  cK-galactan,  so  named  by  Mtintz  (199),  the  first  to  identify 
galactan  as  an  anhydride  of  galactose;  it  composes  42  per  cent  of 
luzerne  seeds  and  occurs  also  in  beans,  barley,  and  malt. 

2.  /3-galactan,  isolated  from  the  Hme  residues  in  the  sugar  beet 
industry  by  Lippmann   (192). 

3.  7-galactan,  first  isolated  from  Chinese  moss  {Sphaerococcus 
lichenoides)  by  Payen  (262),  in  1859,  and  by  him  called  "gelose." 
He  also  identified  it  in  agar-agar^  {Gelidimn  corneum)  and  other  algae. 
The  carbohydrates  of  agar-agar  were  again  studied  by  Reichardt  in 
1876,  who  obtained  a  substance  of  the  formula  Ci2H220n  and  con- 
sidered it  identical  with  the  "pararabin"  which  he  foiuid  in  carrots 
and  beets.=  In  1881  and  1882,  Greenish  (180,  181)  investigated  the 
carbohydrates  of  Fucus  amylaceus  (Ceylon  agar-agar)  and  obtained 
on  hydrolysis  a  sugar-yielding  mucic  acid  (galactose).  From  Sphaero- 
coccus lichenoides  he  also  obtained  a  substance  resembling  Payen's 
"gelose."  In  1884,  Bauer  (169)  showed  that  agar-agar  yields  galac- 
tose; and  in  1905,  Konig  and  Bettels  (190)  gave  the  following  per- 
centage composition  of  Japanese  agar-agar  from  Gelidium: 

Per  cent.  Per  cent- 

Galactans 33         Ash 3.5 

Water 20         Pentosans 3.1 

Protein 2.6      Crude  fiber _..._. 0.4 

^The  term  agar-agar  is  applied  to  the  hot  water  extract  of  various  red  algae, 
mainly  species  of  Gelidium. 
^See  Pentosans,  p.  268. 


Nutrition  Investigations.  283 

Another  species  of  marine  algae  in  which  galactan  has  been  fully 
identified,  is  Chondrus  crispus  (Irish  moss).  This  is  also  a  red  alga. 
C.  Schmidt  (210)  first  examined  it,  in  1844;  he  demonstrated  that 
the  gelatinizing  substance  was  a  carbohydrate  and  yielded  sugar  on 
hydrolysis.  Fliickmger  and  Mayer  (178),  m  1868,  discovered  that 
the  water  extract  of  this  alga  yielded  considerable  mucic  acid.  In 
1875,  Bente  (171)  obtained  levulinic  acid  from  the  products  of  its 
hydrolysis,  and  in  1876,  reported  that  it  jdelded  a  non-crystallizing 
syrup  (172).  The  first  quantitative  analysis  was  made  by  Hadike, 
Bauer  and  Tollens  (185),  who  showed  that  the  water  extract  yielded 
mucic  acid  corresponding  to  about  25  per  cent  of  galactan.  Sebor 
(220),  in  1900,  foimd  in  the  products  of  hydrolysis,  glucose,  fructose 
and  a  small  quantity  of  pentose.  These  observations  were  verified 
by  Miither  (200)  in  1903,  who  further  identified  the  galactose  as  a 
(^-galactose.  From  the  large  yield  of  mucic  acid,  the  water  extract 
of  Chondrus  may  therefore  be  regarded  as  chiefly  galactan,  together 
with  some  dextran  and  levulan,  and  a  very  little  pentosan;  groups 
which,  according  to  Hadike,  Bauer  and  Tollens  (185),  may  be  partly 
or  entirely  bound  into  ester-like  compounds. 

Examples  of  galactans  occurring  in  combination,  or  close  associa- 
tion with  other  hemicelluloses  are  numerous.  Lupeose,  from  luzerne 
seeds,  originally  called  |3-galactan,  yields  50  per  cent  galactose  and  50 
per  cent  fructose  (214).  The  tuberous  roots  of  Stachys  tuberifera 
contain  a  soluble  crystallizable  carbohydrate  yielding  37  per  cent 
mucic  acid,  along  with  an  miidentified  sugar  (225).  Para-galactan 
{galacto-araban)  forms  a  large  proportion  of  the  reserve  material  of 
many  seeds.i  Rothenf  usser  (204)  finds  that  the  mucilaginous  extract  of 
flaxseed  yields  equal  parts  of  pentosans  and  hexosans,  the  latter  being 
mainly  galactose.  Galactans  and  pentosans,  as  already  indicated,^ 
occur  together  in  many  lichens  and  algae,  and  also  in  the  pectins.^ 
Herissey  (187)  has  shown  that  the  "galactine"  of  Miintz  (199)  yields 
equal  parts  of  galactose  and  mannose.  Galacto-mannans  also  fre- 
quently occur  in  the  reserve  material  of  seeds,  as  in  those  of  the  date 
and  other  species  of  palm,  and  in  coffee  beans;  in  the  American  honey 

iCf.  Schiilze  (215),  Schulze,  Steiger  and  Maxwell  (217),  Schulze  and  Castoro 
(218),  Castoro  (176),  and  Goret  (179).  Also  Schulze  and  Godet,  Zeitschrift  fiir 
physiologische  Chemie,  V.  61,  p.  279,  for  a  very  complete  review  of  the  work  of 
Schulze  and  his  pupils. 

2See  Chemical  Nature  of  Lichens  and  Algae:-  Konig  and  Bettels  (8),  Escombe  (6), 
K.  MuUer  (11),  Ulander  (26). 

^Cf.  Pentosans,  p.  268. 


284  Mary  Davies  Swarlz, 

locust  {Gleditschia  triacanthus),  Goret  (179)  found  the  albumen  to 
jdeld  66-70  per  cent  galactose  and  22-23  per  cent  mannose;  he  has 
shown,  in  fact,  that  the  carbohydrate  reserve  of  almost  all  seeds  with 
horny  albumen  consists  largely  of  a  mixture  of  mannans  and  galac- 
tans.i 

GALACTANASES  IN  THE  VEGETABLE  KINGDOM. 

The  hydrolysis  of  the  paragalactan  of  lupine  seeds  during  germina- 
tion was  first  observed  by  Schulze  and  his  co-workers.  That  ordi- 
nary diastatic  enzymes  do  not  form  sugar  from  the  para-galactan  of 
Lupinus  hirsukis  was  demonstrated  by  Schiilze  and  Castoro  (218). 
Ptyalin,  pancreatin,  malt  diastase  and"taka"  diastase,  will,  however, 
in  the  course  of  5  or  6  days'  digestion  at  35^0°  C.  render  this  carbo- 
hydrate soluble  in  water  to  the  following  extent; 

Per  cent.  Per  cent. 

Malt  diastase 38     Ptyalin 40 

Taka  diastase 35      Pancreatin 15 

Griiss  (184)  has  made  exhaustive  microchemical  investigations  upon 
the  germinating  date  endosperm,  in  which  he  has  been  able  to  observe 
the  solution  of  the  galactans  by  enzymes  developed  during  germina- 
tion. Bourquelot  and  Herissey  (174)  find  a  soluble  enzyme  hydrolyz- 
ing  galactan,^  produced  by  the  germinating  embryos  of  the  seeds  of 
the  carob,  Nux  vomica,  fenugrec  and  luzerne.  Shellenberg  (208), 
studying  the  action  of  moulds  on  hemicelliiloses,  foimd  at  least  four 
different  ferments  showing  considerable  specificity  in  their  action; 
seeds  of  Lupinus  hirsutus  (containing  paragalactan)  were  attacked 
by  most  of  these  moulds  {Mucor  neglectus,  Mucor  piriforme,  Rhizopus 
nigricans,  Thamnidium  elegans,  Penicillium  glaucum).  Similarly, 
Herissey  (187)  found  galactose  produced  from  manno-galactans  by 
Aspergillus  niger  and  Aspergillus  fuscus;  Saiki  (205)  obtained  sugar 
from  Irish  moss  by  digesting  it  with  inulase  prepared  from  Aspergillus 
niger  and  Penicillium  glaucum;  and  with  "taka"  diastase  prepared 
from  another  mould,  Eurolium  oryzae. 

Little  is  known  of  the  action  of  bacteria  upon  galactans.  Gran 
(182)  found  sugar  produced  from  agar-agar  by  Bacillus  gelaticus, 
through  the  action  of  an  enzyme  which  he  calls  " gelase."     Saiki  (105), 

^Cf.  Mannans,  p.  283;  for  a  further  discussion  of  the  occurrence  of  Galactans  see 
V.  Lippmann,  Chemie  der  Zuckerarten,  Vol.  I,  pp.  686-697. 
2Cf.  Mannans,  p.  284. 


Nutrition  Investigations.  285 

in  experiments  with  B.  coli  communis,  on  culture  media  containing 
different  kinds  of  comminuted  seaweed,  found  a  slight  gas  production 
in  one  culture,  in  media  with  agar-agar  and  Irish  moss. 

GALACTANASES   IN   THE   ANIMAL   KINGDOM. 

The  only  discovered  instance  of  a  galactanase  in  lower  animals  is 
cited  by  Bierry  and  Giaja  (173),  who  found  that  the  hepato-pan- 
creatic  juice  of  Helix  pomatia  produced  galactose  from  extracts  of 
carob  seeds  {Ceratonia  siliqiia) ;  later  experiments  upon  agar-agar. 
with  extracts  from  a  number  of  crustaceans  {Astacus  fiuviatilis 
Rondel,  Homarus  vulgaris  Bel.,  Maja  squinado  Rondel.,  Carcinus 
moenas  L.,  and  Platycarcinus  pagarus  L.)  were  entirely  negative;  the 
galactans  of  luzerne  and  f  enugrec  were  attacked  with  difficulty  by  the 
extract  from  Astacus.  Strauss  (221)  could  find  no  enzyme  attack- 
ing agar-agar,  in  the  larvae  and  puppae  of  various  species  of  Lepidop- 
tera  and  Diptera. 

No  galactanases  have  been  found  in  higher  animals.  Bierry  and 
Giaja  (173),  using  extracts  of  luzerne  seeds,  got  negative  results 
with  digestive  juices  of  dogs  and  rabbits,  andSawamura  (207)  ob- 
tained similar  results  with  extracts  of  different  sections  of  the  alimen- 
tary canal  of  swine  and  horses.  Saiki  (205)  found  saHva,  pancreatic, 
and  intestinal  juices  unable  to  hydrolyze  Irish  moss, 

DIGESTION  AND  UTILIZATION  OF  GALACTAN  BY  ANIMALS  AND  MAN. 

The  first  study  of  the  digestibility  of  galactans  in  higher  animals 
was  made  in  1903,  by  Lindsey  (191).  Alsike  clover-seed,  containing 
8  per  cent  galactan,  was  fed  in  connection  with  hay,  the  digestibility 
of  which  had  been  previously  determined;  from  analyses  of  food  and 
faeces,  the  galactan  in  the  hay  (1.72  per  cent)  was  found  to  be  75  per 
cent  digestible,  and  that  in  the  clover  95.78  per  cent  digestible. 
Saiki  (205)  fed  agar-agar  and  Irish  moss  to  dogs  and  recovered  a  large 
part  in  the  faeces,  as  shown  by  the  increased  amoimt  of  carbohydrate 
excreted.  Lohrisch  (194)  fed  dogs  and  rabbits  agar-agar  in  its  usual 
form,  and  also  "  soluble-agar "  prepared  from  ordinary  agar  by  Dr. 
Karl  Dieterich  of  Dresden,  Director  of  the  Helfenberg  Chemical  Fac- 
tory. This  product  seems  to  be  partially  hydrolyzed  in  its  prepara- 
tion, since  it  is  not  only  readily  soluble  in  water,  but  has  slight  reduc- 
ing action;  it  yields  on  boiling  with  Fehling's  solution,  3.5-4:.l  per 
cent  sugar,  and  ff  a  watery  solution  is  allowed  to  stand  18   hours  at 


286 


Mary  Davies  Swartz, 


37°  C,  it  is  further  hydrolyzed  and  yields  then  16.9-20.4  per  cent  sugar. 
The  results  of  Lohrisch's  experiments  appear  in  the  folowing  table: 


ANIMAL. 

FOOD. 

HEIUCELLULOSE 

EQUIVALENT  OF   AGAR 

FED. 

HEinCELL- 

UtOSE 
EXCRETED. 

HEMICELL- 

UI.OSE 
DIGESTED. 

Rabbit: 

Rabbit  II 

Ordinary  agar 
Ordinary  agar 
Soluble  agar 
(given  in  9  days) 
Same  as  III 

18.77  =     14.48 

11.8  =      9.11 

95.9  =     65.02 

53.0     =     35.9 

7.1 

4.71 
14.2 

11.7 

Per  cent. 

50.9 

48.3 

Rabbit  III 

Dog 

78.1 
67.3 

Lohrisch  (194)  has  also  studied  the  utilization  of  agar-agar  in  starv- 
ing herbiVora.  In  two  experiments,  rabbits  starved  for  two  days 
were  fed  ordinary  agar  as  long  as  they  would  eat  it,  other  animals  of 
the  same  weight  being  kept  in  starvation  as  controls ;  in  a  third  expe- 
riment,"  soluble  agar"  was  fed.  Urine  and  faeces  were  collected  and 
analyzed.  Of  the  ordinary  agar,  about  50  per  cent  was  excreted  in 
the  faeces;  of  "  soluble  agar,"  about  25  per  cent.  No  positive  evidence 
of  any  change  in  nitrogen  excretion  attributable  to  the  agar  fed,  can 
be  drawn  from  the  protocols.  One  animal  died  through  accident, 
another  survived  its  control  but  one  day,  and  the  third,  in  spite  of  its 
apparently  good  digestion  of  the  "soluble  agar,"  died  a  week  before 
its  control. 

In  the  case  of  rabbits  made  diabetic  with  phlorhizin  and  then  fed 
20^0  grams  of  both  ordinary  and  soluble  agar,  Lohrisch  (194)  found 
that  the  D :  N  ratio  remained  fairly  constant  throughout  each  experi- 
ment, showing  no  marked  increase  in  sugar  excretion.  We  see,  there- 
fore, no  grounds  for  assuming  that  agar-agar  {galactan)  forms  glycogen 
in  rabbits. 

The  first  studies  on  the  utilization  of  galactan  by  man  were  made 
by  Saiki  (205)  (1906).  In  feeding  experiments  in  which  various  car- 
bohydrates were  at  different  times  added  to  a  uniform  diet,  consisting 
of  513  grams  beefsteak,  500-600  grams  bread,  40  grams  sugar,  31 
grams  butter,  2  eggs  and  2  apples  —  a  diet  on  which  over  98  per  cent 
of  the  carbohydrates  were  digested,  he  obtained  the  following  results: 


Nutrition  Investigations. 


287 


STJBSTANCE    ADDED    TO    DIET. 


EQUIVALENT  OS 

SUBSTANCE  IN 

DEXTROSE. 


CARBOHYDRATES 
IN  FAECES  CAL- 
CULATED  AS 
DEXTROSE. 


HEMICELLULOSE 
DIGESTED. 


20  grams  agar .  . . 
24  grams  agar .  . . 
40  grams  wakame 
45  grams  kom.bu. 


Crams. 

10 
12 
4.7 
11.4 


Grams. 
9.2 
8.8 
3.4 
2.5 


Per  cent. 

8 
27 
28 
78 


Lohrisch  has  also  studied  the  digestibility  of  "soluble  agar"  in 
man.  Sometimes  it  is  not  well  borne,  especially  if  given  in  quanti- 
ties over  50-60  grams  per  day  and  causes  gas  formation,  diarrhoea, 
and  other  intestinal  disturbances;  in  other  cases,  large  amounts  (100 
grams  per  day)  cause  no  unpleasant  symptoms  whatever.  The  agar 
was  dissolved  in  some  beverage,  and  the  diet  was  otherwise  carbohy- 
drate-free.    Some  of  the  results  are  shown  in  the  following  table  (194) : 


DURATION  OE 
EXPERIMENT. 

AMOUNT 

DIGESTED. 

HEMICELL- 
ULOSE 
DIGESTED. 

HEMICELL- 

NO. 

As  Air  Dry 
Soluble  Agar. 

As  Hemicel- 
lulose. 

HEMiCKJLLULOSE 
EXCRETED. 

ULOSE 
DIGESTED. 

Grams. 

Grams. 

GrOOTS. 

Grama. 

Per  cent. 

1 

1  day 

100 

61.9 

46.06 

15.84 

25.6 

2 

1  day 

100 

61.9 

39.1 

22.8 

36.8 

3 

3  days 

235 

145.4 

90.5 

54.9 

37.7 

4 

3  days 

240 

148.5 

40.8 

107.7 

72.5 

5 

1  day 

100 

61.9 

25.4 

36.5 

58.9 

6 

1  day 

110 

67.8 

23.4 

44.4 

65.5 

No.  4  was  a  case  of  chronic  constipation;  the  high  percentage  of  hemi- 
cellulose  digested  is  in  accordance  with  the  observations  of  Lohrisch 
(193)  and  Pletnew  (203),  on  the  extraordinarily  good  utilization  of  all 
foodstuffs  in  chronic  constipation.  Two  of  these  experiments  were  on 
diabetics,  and  showed  that  the  18.36  grams  of  "soluble  agar"  ab- 
sorbed per  day  caused  no  increase  of  sugar  in  the  urine,  and  had  no 
noticeable  effect  on  nitrogen  metaboUsm. 

From  these  experiments,  we  see  that  ordinary  agar  is  digestible  to  a 
very  small  extent,  and  that  even  when  changed  to  an  easily  hydro- 
lyzed  form,  it  is  only  digested  to  about  50  per  cent.  Is  the  part 
digested  absorbed  and  utilized  as  galactose?    The  recent  exhaustive 


288 


Mary  Davies  Swartz, 


discussion  of  the  behavior  of  galactose  in  the  animal  body  by  Brasch 
(175)  renders  any  details  on  the  utilization  of  this  sugar  unnecessary. 
Hofmeister  (188)  showed  that  of  all  sugars  it  is  most  readily  excreted. 
That  galactose  can  form  glycogen  in  dogs  and  rabbits,  has  been  shown 
by  Weinland  (226),  Kausch  and  Socin  (189),  Cremer  (177),  Voit  (223), 
Brasch  (175),  and  others.^  Brasch  (175)  has  shown  that  the  assimila- 
tion limits  for  galactose  lie,  for  normal  man,  between  30  and  40  grams, 
while  for  dextrose  they  lie  between  100  and  150  grams.  Voit  (224), 
Sandmeyer  (206), Bauer  (170),  and  others  have  shown  that  galactose, 
even  in  small  amounts  increases  the  sugar  excretion  in  diabetes.  It 
would  seem,  therefore,  that  if  soluble  agar  were  absorbed  as  sugar,  it 
would  increase  the  sugar  output  in  the  urine.  To  throw  some  light 
on  this  problem  Lohrisch  (194)  has  conducted  three  respiration  ex- 
periments on  men  after  ingestion  of  100-110  grams  of  soluble  agar,  of 
which,  on  the  average,  about  63  per  cent  was  absorbed.  The  changes 
in  the  respiratory  quotient  are  shown  in  the  following  table: 


Respiratory  Quotient. 


NUMBER  OF  HOURS  AFTER  INGESTION  OF  SOLUBLE  AGAR. 


NO. 

IN  FASTING. 

1          i            2 

3 

4 

5 

6 

7 

I 

II 

III 

0.768 
0.786 
0.739 

0.768 

0.766 

0.835 
0.794 
0.815 

0.860 
0.825 
0.800 

0.770 
0.767 
0.774 

0.735 

0.724 
0.714 

IN  FASTING. 

NUMBER  OF  HO 

URS  AFTER  INGESTION   OF 

SOLUBLE  AGAR. 

NO. 

8 

9 

10 

11 

12 

13 

I 

II 

III 

0.768 
0.786 
0.739 

0.693 

0.730 

0.703 

0.618 

0.669 

The  distinct  rise  in  the  respiratory  quotient  in  the  fourth  hour 
(beginning  in  the  third  hour  in  Experiment  I)  would  indicate  that  car- 
bohydrate was  being  oxidized,  which  in  this  case  must  come  from  the 
agar.  The  low  value  in  the  later  hours  seems  due  to  the  oxidation  of 
fatty  acids  ;2  that  such  acids  may  be  formed  from  soluble  agar  by 
bacteria,  appears  probable  also  from  the  intestinal  fermentation  pro- 


^Cf .  Magnus-Levy,  Verwerthbarkeit  der  Galactose  in  normalen  Organismus :  Op- 
penheimer's  Handbuch  der  Biochemie  der  Menschen  und  der  Tiere,  Vol.  IV,  p.  379. 
^Cf.  respiration  experiments  described  under  Cellulose. 


Nutrition  Investigations.  289 

duced  when  large  amounts  of  this  preparation  are  taken.  A  slight 
increase  in  acetone  output,  shown  in  the  metabolism  experiments 
with  diabetics,  points  to  the  same  conclusion.  Perhaps,  as  Lohrisch 
suggests,  the  very  slow  digestion  of  the  carbohydrate,  may  enable  the 
organism  to  utUize  the  galactose  formed,  and  accoimt  for  its  non-ex- 
cretion, but  this  reqiiires  further  demonstration. 

According  to  these  experiments  by  Lohrisch,  cellulose  and  the  solu- 
ble galactan  show  Httle  difference  in  their  physiological  behavior. 
Both  can  be  digested  to  about  50  per  cent.  Ordinary  agar,  as  Saiki's 
experiments  show,  is  largely  recovered  in  the  faeces;  in  fact, a  thera- 
peutic practice  which  has  been  recently  estabHshed  is  based  upon  the 
recognized  indigestibiUty  of  agar,  namely,  its  employment  as  a  remedy 
in  cases  of  chronic  constipation.  It  is  especially  valuable,  as  Mendel 
(196)  points  out,  in  those  cases  where  the  difficulty  is  due  to  an  ex- 
tremely complete  digestion  and  absorption  of  all  foodstuffs  from  the 
alimentary  tract,  which  causes  the  formation  of  dry,  hard  faecal 
masses  {scyhalla)  difficult  to  evacuate.  The  agar,  remaining  imdigested 
and  retaining  a  high  percentage  of  water,  gives  bulk  and  softness  to 
the  faeces,  and  facilitates  their  daUy  elimination.  Being  resistant 
towards  bacterial  action,  it  causes  neither  gas  formation  nor  produc- 
tion of  harmful  decomposition  products.  According  to  A.  Schmidt 
(209),  it  can  be  advantageously  taken  in  quantities  up  to  25  grams  per 
day,  part  with  the  breakfast  cereal,  and  part  with  sauce  or  cream,  at 
another  meal.  In  view  of  such  facts  as  these,  we  are  hardly  prepared 
to  agree  with  Lohrisch,  that '  Cellulose  and  Hemicelluloses  are  readily 
digested. ' 

Occurrence  and  Nature  oe  Mannans. 

•  As  widely  diversified  in  origin  and  character  as  the  galactans,  and 
very  intimately  associated  with  them  are  the  Mannans.  They  show 
all  possible  degrees  of  solubiHty,  from  the  readily  soluble  mucilage 
found  in  certain  legumes,  to  the  completely  insoluble  "  reserve-ceUu- 
lose,"  which  forms  the  horny  albumen  in  such  seeds  as  the  date,  and 
which  was  long  confused  with  true  cellulose. 

A  few  examples  will  serve  to  show  the  diverse  places  in  which  man- 
nans may  be  found.  They  occur  in  yeast  :i  (258)  in  algae,  as  Por- 
phyra  laciniata;  (278)  in  moulds,  as  Penicillium  glaucum;  (285)  in  the 
leaves  and  roots  of  the  Japanese  plant,  Conophallus  konjaku  (280) ; 
in  the  bark  and  wood  of  many  American  trees  (272) . 

iFor  further  discussion  see  v.Lippmann,  Chemie  der  Zuckerarten,  Vol.1,  pp.  641- 
649,  and  Czapek,  Biochemie  der  Pflanzen,  pp.  325-329. 


290  Mary  Davles  Swartz, 

The  most  extensive  study  has  been  given  to  the  mannans  of  various 
seeds,  in  which,  as  already  shown, ^  mannans  and  galactans  seem  al- 
most invariably  to  occur  together.  The  seeds  of  the  carob  tree  {Ce~ 
ratonia  siliqud)  contain  a  hemicellulose  originally  called  "caruban" 
by  Efiront  (241)  (1897),  but  shown  by  van  Ekenstein  (282)  to  yield 
mannose,  and  by  Bourquelot  and  Herissey  (232)  (1899),  (^-galactose. 
The  first  elaborate  studies  of  "reserve-cellulose"  were  made  by  Reiss 
(264),  who  showed  that  the  horny  albumen  of  the  seeds  of  Phytelepas 
macrocarpa,  Phoenix  dactylifera  and  other  species  of  palm,  Allium 
cepa,  Asparagus  officinalis,  Iris  pseudacorus,  Strychnos  nux  vomica 
and  Caffea  arabica,  differed  chemically  from  true  cellulose  in  their 
color  reactions,  in  the  ease  with  which  they  can  be  hydrolyzed,  and  in 
yielding,  instead  of  dextrose,  a  sugar  which  he  called  "seminose," 
but  which  proved  to  be  identical  with  Fischer  and  Hirschberger's  (242) 
previously  described  mannose. 

Mannan  also  occurs  richly  in  the  tubers  of  the  many  species  of  Or- 
chis and  Eulophia  which  are  the  source  of  commercial  salep.  On  ex- 
traction with  water,  they  yield  a  mucilaginous  extract  which  was 
first  studied  by  C.  Schmidt  (270)  in  1844,  and  called  by  him  "salep- 
bassorin";  on  hydrolysis  with  dilute  sulphuric  acid  he  obtained,  be- 
s'des  some  gummy  substance  and  cellulose,  a  fermentable  sugar 
which  he  thought  to  be  dextrose.  Mulder  (259)  considered  the  salep 
mucilage  a  mixture  of  starch  and  gum  or  pectin  acids,  while  Franck 
(243)  thought  it  a  modification  of  cellulose,  and  Girand  (248)  a  trans- 
formation of  a  starchy  substance  into  a  variety  of  dextrin  swelling  in 
water.  Pohl  (263)  by  precipitation  with  neutral  salts,  distinguished 
an  " a-Schleim "  and  a  "/3-Schleim. "  According  to  Thamm  (276) ,  who 
has  made  the  most  recent  investigations,  ''a-Schleim"  does  not  occur 
in  German  salep.  ToUens  and  Gans  (277)  showed  that  on  hydrolysis, 
besides  dextrose,  mannose  or,  as  they  called  it,  "  isomanitose "  was 
formed,  but  this  was  shown  by  Fischer  and  Hirschberger  (242)  to  be 
identical  with  ^-marmose.  Thamm  (276)  and  Hilger  (254)  have  shown 
conclusively,  that  the  starch-free  water  extract  contains  an  anhydride 
of  mannose  only. 

A  very  resistant  type  of  mannan  occurring  in  some  plants,  has  been 
designated  as  manno-cellulose  by  Schulze  (273).  Bertrand  (227) 
finds  it  taking  the  place  of  xylan  in  the  woody  tissues  of  gymnosperms. 


^Cf .  Schulze  and  his  coworkers,  and  Goret,  under  Galactans.     Also  Schulze  and 
Godet,  Zeitschrift  fiir  physiologische  Chemie,  V.  61,  p.  279,  for  a  very  complete 


Nutrition  Investigations.  291 

MANNANASES   IN   THE   VEGETABLE   KINGDOM. 

There  is  very  little  literature  concerning  the  action  of  bacteria  upon 
mannans.  Sawamura  (267)  observed  that  extracts  of  Hydrangea  pa- 
niculata,  used  in  the  manufacture  of  Japanese  paper,  which  contain 
mannan  (along  with  galactan  and  araban) ,  became  liquefied  on  stand- 
ing. In  bacteriological  studies  with  extracts  of  this  plant,  and  of 
roots  of  Conophallus  konjaku,  he  found  that  only  B.  mesentericus  vul- 
gatus  dissolved  these  mannans.  The  action  was  greatly  facilitated, 
and  sugar  formation  increased  if  a  certain  wild  yeast,  in  itself  inactive, 
were  present.  Traces  of  a  similar  enzyme  seem  to  occur  in  B.  prodi- 
giosus. 

In  his  studies  of  the  action  of  moulds  on  hemicelluloses,  Schellen- 
berg  (269)  found  that  the  seeds  of  Ruscus  aculeata,  which  yield  almost 
exclusively  mannose  (237-240),  were  attacked  only  by  Penicillium 
glaucum.  Herissey  (253),  using  pure  cultures  and  water  extracts  of 
cultures  of  Aspergillus  niger  (grown  on  media  rich  in  mannose  and  ga- 
lactose to  incite  the  development  of  mannanase  and  galactanase),  with 
suitable  antiseptics  and  controls,  obtained  mannose  —  and  galactose 
—  from  seeds  of  Ceratonia  siliqua  and  Gleditschia  triacanthus,  and  an 
abundant  jdeld  of  mannose  from  salep;  similar  results  were  obtained 
with  Aspergillus  f us cus. 

As  early  as  1862,  Sachs  (266)  observed  the  change  of  the  thickened 
cell-walls  of  the  date  endosperm  into  sugar  during  germination.  The 
cytases  producing  this  change  in  'reserve-cellulose'  were  later  care- 
fully investigated  by  Reiss  (264),  Brown  and  Morris  (230),  Newcombe 
(261),  Griiss  (251),  and  others.  Still  more  recently,  Bourquelot  and 
Herissey  have  made  many  studies  on  the  specific  characteristics  of 
these  plant  enzymes.  An  exhaustive  review  of  the  literature  on  man- 
nans and  the  action  of  enzymes  upon  them  has  been  published  by 
Herissey  (253),  consequently  this  subject  will  only  be  reviewed  very 
briefly  here. 

Gruss  (251)  has  demonstrated  that  the  solution  of  the  date  embryo 
(Phoenix  dactylifera)  is  due  to  a  ferment,  the  product  of  whose  activ- 
ity is  galactan  and  mannose.  Effront  (241)  (in  1897)  attributed  the 
solution  of  the  albumen  of  carob  seeds  (called  by  him  caruban)  to  a 
"caroubinase,"  but  thought  that  the  product  of  its  activity  was  not 
identical  with  the  products  of  hydrolysis;  in  1899,  however,  Bourque- 
lot and  Herissey  (233)  showed  the  possibiUty  of  obtaining  mannose 
by  the  action  of  a  soluble  ferment  derived  from  these  seeds,  which 
they  called  "seminase."     Shortly  afterwards,  a  similar  enzyme  was 


292  Mary  Davies  Swartz, 

isolated  by  them  from  the  seeds  of  Phoenix  canariensis.  Herissey 
(253)  has  been  able  to  show  that  seeds  of  such  legumes  as  luzerne, 
fenugrec,  and  common  genet  have,  at  least  at  the  time  of  germination, 
ferments  capable  of  transforming  mannans — and  galactans — into 
their  corresponding  sugars.  Experiments  in  vitro  show  that  they  are 
not  limited  to  action  upon  the  seeds  by  whose  embryos  they  are  pro- 
duced; but  act  on  the  reserve-cellulose  of  seeds  from  very  distinct 
groups  of  plants.  However,  the  luzerne  ferment  does  not  digest  all 
mannans  and  galactans;  it  will  hydrolyze  the  mannans  of  the  tubers 
of  the  Orchis  family  (and  commercial  salep  prepared  from  them),  but 
not  those  of  the  albumen  of  palm  seeds. 

Griiss  (251)  has  also  shown  that  the  enzyme  of  the  date  endosperm 
hydrolyzes  starch,  although  this  does  not  occur  in  the  date  seed,  and 
that  malt  diastase  works  on  a-mannan  (the  soluble  mannan  of  date 
seeds,  according  to  Griiss)  which  does  not  occur  in  the  barley  endo- 
sperm. Griiss  considers  diastatic  enzymes  a  group  working  not  only 
on  starch,  but  also  on  hemicelluloses.  Herissey  thinks  that  diastase 
and  seminase  are  foimd  together  in  varying  proportions  in  barley, 
legumes,  carob  seeds,  etc.,  and  that  neither  is  a  simple  ferment,  but  a 
"superposition  de  ferments,"  and  defines  "seminase"  as  a  "ferment 
or  group  of  soluble  ferments,  causing  the  transformation  of  the  car- 
bohydrates of  horny  albumens  of  the  seeds  of  Leguminosae  into  as- 
similable sugars."  Gatin  (247)  has  made  further  researches  upon  the 
natiire  of  seminase,  and  states  that  during  the  germination  of  certain 
seeds  whose  reserve  is  in  the  form  of  mannan,  the  presence  of  mannose 
is  exceptional,  but  dextrose  occurs  in  abundance.  This  phenomenon 
he  attributes  to  a  "  manno-isomerase,"  which  transforms  the  mannose, 
as  fast  as  formed  by  the  seminase,  into  dextrose.  Experiments  in 
vitro  seem  to  indicate  that  this  is  a  soluble  ferment. 

MANNANASES  IN   THE  ANIMAL   KINGDOM. 

There  are  only  a  few  instances  on  record  of  mansases  occurring  in 
lower  animals.  Bierry  and  Giaja  (228,  229)  found  that  the  hepato- 
pancreatic  juice  of  Helix  pomatia  was  capable  of  producing  mannose 
from  extracts  of  carob  seeds  and  salep;  that  of  Astacus  fluviatilis,  Ho- 
marus  vulgaris,  and  Maja  squinado,  from  the  ivory  nut  {Phytelepas  ma- 
crocarpa),  the  two  latter  hydrolyzing  it  at  ordinary  room  temperature. 
On  the  other  hand,  the  mannans  of  fenugrec  and  luzerne  were  hydro- 
lyzed  with  difi&culty,  or  not  at  all,  by  very  pure  gastro-intestinal 
juice.     No  mannanase  was  found  by  Strauss  (275)  in  the  larvae  and 


Nutrition  Investigations  293 

puppae  of  Lepidoptera  and  Diptera.  Similar  negative  results  have 
been  obtained  with  the  digestive  enzymes  of  higher  animals.  Kino- 
shita  (257)  f  oiind  that  emulsin  and  invertin  did  not  hydrolyze  the  man- 
nans  of  Conophallus  konjaku  and  Gatin  (245,  246)  tried  the  blood  of 
rabbits,  chicken  serimi,  the  pancreatic  juice  of  dogs,  the  macerated 
intestines  and  pancreas  of  chickens  and  cattle,  upon  salep  and  carob 
seeds  with  negative  results;  on  the  other  hand,  Sawamiira  (268)  re- 
ports a  mannanase  in  the  extracts  from  different  sections  of  the  ali- 
mentary tract  of  swine  and  horses. 

DIGESTION  AND   UTILIZATION   BY  ANIMALS   AND  MAN. 

There  are  also  very  few  records  in  the  literature  of  feeding  experi- 
ments with  mannans.  In  a  paper  in  the  Zeitschrift  fiir  Biologic,  Voit 
(283)  in  1874^  described  one  by  Hauber,  who  fed  a  medium  sized  dog 
390  grams  of  dry  salep  powder  in  the  course  of  eight  days.  The  faeces 
of  the  feeding  period  were  roughly  marked  ofi,  and  Hauber  reported 
no  imchanged  salep  present  in  them,  because  there  was  no  swelling 
in  water  as  with  the  original  powder.  Calculations  based  on  the  yield 
of  sugar  from  the  faeces  on  hydrolysis  showed  that  at  least  50  per  cent 
of  the  salep  was  absorbed.  This  seems  to  have  been  a  very  crude  ex- 
periment, and  cannot  be  considered  of  convincing  value. 

In  1879,  Weiske  (284)  fed  carob-beans  {Ceratonia  siligua)  to  sheep, 
along  with  meadow  hay,  and  compared  the  nutritive  value  of  this  ra- 
tion with  one  in  which  the  carob-beans  (210  grams)  were  replaced  by 
an  equivalent  weight  of  starch,  sugar  and  protein  (from  crushed  peas). 
The  coefficients  of  digestibility  and  nitrogen  balance  were  so  nearly 
the  same  on  the  two  rations,  that  Weiske  pronounced  "Johannis- 
brod"  (carob  beans)  an  acceptable  and  digestible  feed  for  sheep. 

In  1890,  Schuster  and  Liebscher  (274)  tried  feeding  the  sawdust  of 
ivory  nut  {Phytelepas  macrocarpa)  to  sheep,  having  previously  found 
that  it  had  a  favorable  effect  on  cattle.  Merino  sheep  gained  consider- 
able fat  when  fed  oat  straw  and  vetch  fodder,  plus  ivory  nut  sawdust 
furnishing  50  per  cent  of  the  digestible  carbohydrates.  The  ration, 
exclusive  of  the  ivory  nut,  did  not  3deld  enough  energy  for  such  a  re- 
siilt  to  be  possible,  hence  the  latter  must  have  been  utiHzed.  The 
coefficient  of  digestibiUty,  both  for  the  nitrogen-free  extract  and  crude 
fiber  of  this  material,  was  at  the  same  time  shown  by  Niebling  (262) 
to  be  82  per  cent  for  sheep. 


^This  paper  reviews  the  early  literature  on  gums. 


294  Mary  Davies  Swartz, 

From  these  experiments,  mannan  would  seem  to  be  well  utilized  by 
herbivora.  The  only  experimental  data  regarding  the  nutritive  value 
of  mannans  to  man,  are  cited  by  Oshima  (15)  from  work  by  Kano  and 
lishima  (255),  who  found  the  coefi&cient  of  digestibility  of  konjaku  82 
per  cent  (prepared from Cowo/>/?a^?M5  konjaku).  Further  investigations 
seem  highly  desirable,  in  view  of  the  fact  that  in  certain  regions  food 
stuffs  like  salep  and  konjaku,  consisting  of  almost  pure  mannan,  are 
among  the  chief  articles  of  the  poor  man's  diet.  It  is  also  a  question 
whether  the  nutritive  value  of  bark,  especially  of  coniferous  trees,  is 
due  to  mannan  present.  According  to  Dillingham  (239)  the  quantity 
of  mannan  present  does  not  justify  such  an  assumption,  aside  from 
the  question  of  its  digestibility. 

We  have  finally  to  inquire  whether  mannan  can  be  hydrolyzed 
within  the  organism,  and  if  so,  whether  the  mannose  produced  can  be 
retained  and  form  glycogen.  From  the  literature  on  the  subject,  it 
appears  that  mannose  is  well  utilized  by  rabbits,  dogs  and  men.  Ac- 
cording to  Neuberg  and  Mayer  (260),  the  d-iorvo.  is  better  utilized 
than  the  I-  or  i-form.  Mannose  is  readily  converted  to  dextrose  in 
the  organism;  thus  Neuberg  and  Mayer  found  that  a  rabbit,  receiv- 
ing 10  grams  of  /-mannose  per  os,  excreted  1  gram  /-mannose  and  4-5 
grams  /-glucose;  10  grams  of  c?-mannose  given  rabbits  per  os,  or  sub- 
cutaneously,  were  almost  completely  oxidized.  Rabbits  fed  30  grams 
{^-mannose  by  Cremer  (238)  excreted  3-4  grams  in  the  urine,  and  dogs 
given  20  grams  by  Rosenfeld  (265),  excreted  over  4  grams.  This  is 
somewhat  more  than  would  be  excreted  on  giving  equally  large  quan- 
tities of  dextrose  or  levulose.  Cremer  (238)  found  no  sugar  in  the 
urine  of  a  man  after  feeding  3-12  grams  of  mannose. 

That  mannose  can  act  as  a  glycogen  former  in  rabbits,  has  been 
demonstrated  by  Cremer  (238)  and  also  by  Rosenfeld  (265).  Neu- 
berg and  Mayer  (260)  found  only  a  small  amount  of  glycogen  in  the 
livers  of  starving  rabbits  after  feeding  /-mannose,  but  even  this  form 
is  utilized  to  some  extent.  There  is  good  reason  for  assuming,  there- 
fore, that  if  mannans  can  be  converted  into  mannose  in  the  process 
of  digestion,  they  may  be  considered  as  true  nutrients  for  the  organ- 
ism, the  mannose  being  to  a  high  degree  capable  of  absorption  and  con- 
version into  glycogen. 


Nutrition  Investigations. 


295 


OCCUERENCE  AND  NATURE  OE  LeVULANS. 

A  number  of  polysaccharide  carbohydrates  yielding  levulose  on 
inversion  have  been  described.  They  are  all  levo-rotatory,  more  or 
less  soluble  in  cold  water  and  insoluble  in  alcohol,  and  easily  hydro- 
lyzed  by  dilute  acid,  but  have  not  been  investigated  sufficiently  to 
permit  any  conclusion  to  be  drawn  respecting  their  relation  to  one 
another.  The  most  important  of  these  substances  and  their  sources 
are  shown  in  the  following  table:* 


NAME. 

SOURCE. 

INVESIIGATOR. 

Inulin 

Tubers  of  dahlia,  artichoke,  Jerusalem 
artichoke,   elecampane;    bulbs  of  onion, 
garlic,    narcissus,    hyacinth,    and   tube- 
rose;   flowers,    seed,    etc.,    of    various 
compositae 

Tanret  (321) 

Chevastelon  (291) 

Pseudo-inulin 
Inulenin 
Helianthin 
Synanthrin 

Tubers  of  dahlia,  artichoke,  Jerusalem 
artichoke,  elecampane;  bulbs  of  onion, 
garlic,   narcissus,   hyacinth,    and    tube- 
rose;   flowers,     seed,    etc.,    of    various 
compositae 

Tanret  (321,  322) 

Levulin 

Tubers  of  Helianthus  tuberosus  (Jeru- 
salem artichoke) 

Reidemeister  (314) 
and  others 

Phlein 

Rootstalks  of  Phleimi  praetense  (Tim- 
othy) 

Ekstrand    and    Jo- 
hanson  (296) 

Cerosin 

Unripe  grains 

Tanret  (320) 

Graminin 

Rootstalks    of    various  grasses,    e.g., 
Trisetmn  alpestre 

Ekstrand  and  Johan- 

son  (296) 
Harlay  (301) 

Triticin 

Dracaena  austrahs  and  rubra,     Triti- 
cum  repens  (couch  grass) 

Reidemeister  (314) 

Sinistrin 

Bulbs  of  Scilla  Maritima  (Sea  onions     Schmiedeberg    (318) 
or  squills)                                                          Reidemeister  (314) 

Levulan 

Molasses  in  beet-sugar  industry 

v.  Lippmann  (309) 

'  Cf.  V.  Lippmann,  Chemie  der  Zuckerarten,  Vol.  I,  pp.  795-807. 


296 


Mary  Davies  Swartz 


The  best  known  member  of  this  group  is  inulin,i  closely  associated 
with  which  are  the  four  levulans  described  by  Tanret;  these  seem  to 
be  intermediate  products  between  inulin  and  levulose,  all  having 
greater  solubility  than  inulin,  but  less  levo-rotatory  power.  The 
other  carbohydrates  mentioned  are  also  more  soluble  than  inulin, 
but  have  higher  specific  rotation. 

LEVULANASES  IN  THE  VEGETABLE  KINGDOM. 

Comparatively  few  studies  have  been  made  upon  the  action  of 
enzymes  on  the  levulans,  and  these  have  been  for  the  most  part  lim- 
ited to  invdin.  Certain  micro-organisms  as  B.  Coli  communis  (295), 
Clostridium  pastorianum  (328),  and  several  Schizomycetes,  decom- 
pose inulin,  but  without  any  production  of  sugar.  Yeast,  according 
to  Tanret  (321)  does  not  ordinarily  ferment  it,  but  Lindner  (308) 
asserts  that  certain  forms  of  top  yeast  change  it  readily.  Levulin 
is  fermented  by  yeast,  according  to  Levy  (307),  and  triticin,  in  the 
course  of  four  or  five  days,  according  to  Reidmeister  (314);  but  it 
seems  probable  that  the  first  changes  are  due  to  gradual  hydrolysis 
on  standing  in  water,  or  to  other  organisms. 

The  effect  of  vegetable  enzymes  on  these  carbohydrates,  as  far  as 
they  have  been  studied,  is  shown  in  the  following  table: 


NAME    OF   LEVULAN. 

INVERTIN 
OF   YEAST. 

MALT  DIASTASE. 

"tAKA"  DIASTASE. 

INULASE  OF 
ASPERGILLUS. 

Inulin 

-(8) 
+  (1) 

-(2) 

-(3) 
-(4) 

+  (5) 
-(6) 

-(3) 

+  (7) 

Levulin 

Graminin ... 

+  very 
slowly  (4) 

Triticin 

Sinistrin 

(1)  Levy  (307) 

(2)  Reidemeister  (314) 

(3)  Chittenden  (292) 

(4)  Harlay  (301) 


(5)  Reidemeister  (314) 

(6)  Schmiedeberg  (318) 

(7)  Dean  (293)  and  others 

(8)  Komanos  (303) 


Discovery  of  the  best  known  ferment  for  any  levulan  is  due  to 
Green  (300)  who,  in  1888,  extracted  such  an  enzyme  from  the  tubers 
of  the  Jerusalem  artichoke  {Helianthus  tuber osus),  and  named  it  "in- 


^For  description  and  early  literature  see  Kiliani  (302)  and  Dean  (294). 


Nutrition  Investigations.  297 

ulase."  Subsequently,  Bourquelot  (289)  found  inulase  in  Asper- 
gillus niger  and  Penicillium  glaucum;  and  Chevastelon  (291)  showed 
that  this  enzyme  would  hydrolyze  the  inulin  of  the  monoctyledons. 
Dean  (293)  has  studied  the  properties  of  inulase  exhaustively,  and 
shown  that  in  Aspergillus  and  Penicillium  it  exists  only  as  an  endo- 
enzyme.  Went  (327)  has  found  inulase  also  in  Monilia  sitopkila  and 
other  Amylomyces. 

LEVXTLANASES  IN  ANIMALS. 

The  first  instance  of  an  inulase  in  an  animal  organism  has  been 
cited  by  Strauss  (319).  '  In  1908,  he  reported  studies  on  the  enzymes 
of  seven  species  of  Lepidoptera  and  Diptera,  during  their  various 
stages  of  development  {Euproctis  chrysorrhea,  Ocneria  disparata,  Bom- 
byneustria,  Bomhyx  mori,  Galleria  melonella,  Hyponomenta,  Calliophera 
vomitoria),  but  found  inulase  present  only  in  the  eating  larvae  of 
Bomhyx  mori  and  Hyponomenta.  No  inulase  was  present  in  the  larvae 
of  these  species  after  they  had  ceased  eating,  nor  in  the  pupae  and 
imagines. 

The  results  of  Kobert  (304)  in  1903,  with  extracts  of  May  beetles, 
cross  spiders,  scorpions,  cockroaches,  ascarides,  pupae  of  pine  spiders, 
and  house  flies,  were  entirely  negative;  so  also  have  been  the  experi- 
ments in  vitro  with  digestive  juices  of  higher  animals,  as  shown  by 
table  on  following  page. 


DIGESTION   AND  UTILIZATION   BY  ANIMALS. 

Inulin  is  hydrolyzed  by  very  dilute  acid  (0.05-0.2  per  cent  at  40° 
C,  according  to  Chittenden),  so  that  its  more  or  less  complete 
inversion  by  the  gastric  juice  is  possible,  and  has  led  many  to  believe 
that  in  spite  of  the  negative  results  obtained  with  amylolytic  enzymes 
shown  above,  it  might  be  converted  into  levulose,  and  as  such  be  read- 
ily utilized  by  the  animal  organism.  It  has  therefore  frequently 
been  recommended  for  the  diet  of  diabetics,  who  show  a  special  tol- 
erance for  levulose;  in  fact,  simply  because  inulin  did  not  reappear  in 
the  urine  as  sugar,  when  fed  to  diabetics,  its  utilization  has  been  as- 
sumed by  many,  no  account  being  taken  of  its  possible  reappearance 
in  the  faeces.  This  reappearance  is  well  demonstrated  in  an  experi- 
ment of  Sandmeyer  (317)  in  which,  after  feeding  80  grams  of  inulin 
to  a  diabetic  dog,  over  46  grams  were  recovered  in  the  faeces. 


298 


Mary  Davies  Swartz, 


AUTHORITY. 

DATE. 

SOURCE  OF  ENZYME. 

KIND  OF 
LEVUIAN. 

RESULT. 

Komanos  (303) 

1875 

Saliva 

Inulin 



Pancreatic  juice 

Inulin 

— 

Schmiedeberg  (318).... 

1879 

Saliva 

Sinistrin 

— 

Chittenden  (292) 

1898 

Saliva 

Inulin 

— 

Pancreatic  juice 

Inulin 

— 

Bierry  and  Portier  (288) 

1900 

Macerated   pancreas   and 
intestines  of  dog,    rabbit 

and  seal 

Inulin 

— 

BierryandPortier(288). 

1900 

Macerated  pancreas  and  in- 
testines of  dogs,  rabbits; 
fed  three  months  on  arti- 
chokes to  induce  formation 

of  an  inulase* 

Inulin 

—    - 

Harlay  (301) 

1901 

Saliva 

Graminin 

— 

Bierry  (286) 

1905 

Pancreatic  juice  of  dog 
Pancreatic  juice  of  dog  + 

Inulin 



macerated     intestines    of 

dogs  and  rabbits 

Inulin 

— ^^ 

Bierry  (287) 

1910 

Pancreatic  juice  of  dog  from 
pancreatic  fistula  after  in- 

jection of  secretin 

Inulin 

— 

Same  pancreatic  juice  added 

to  macerated  intestines  of 

dog  and  rabbit,  in  slightly 

acid,  slightly  alkaline  and 

neutral  solutions 

Inulin 

— 

Hepato-pancreatic   juice    of 

Helix  pomatia 

Inulin 

Levulose 

Enzyme  prepared  from  he- 

pato-pancreatic   juice    of 

Helix  pomatia 

Inulin 

Levulose 

Weinland  (326) 

1905 

Extract  of  small  intestine  of 

dog 

Inulin 

— 

'  Cf.  Richaud,  (326). 


Attempts  to  induce  glycogen  formation  in  rabbits  have  not  justi- 
fied the  hopes  of  the  dieto-therapists  in  regard  to  inulin  as  a  food  for 
diabetics.  The  earlier  experiments  were  either  negative  or  open  to 
criticism  on  account  of  faulty  technique.  The  more  discriminating 
work  of  recent  investigators  (Miura  [313] ;  and  Mendel  and  Naka- 
seko  [312]),  has  shown  that  Httle  glycogen  is  formed  from  inulin,  even 
under  the  most  favorable  circumstances.  A  brief  survey  of  the  expe- 
riments in  this  field  is  given  in  the  following  table: 


u     bo 


03   22 

_  '^  ''JO 


<t> 


.a   o   4J 


3 

r3    o 


bo    3 

h     > 


03       CD       O 


fl  a 

S3  :3 


K    p    aj 


o 


60  a 


'■2  s  V 


3  -3 


■Tj     <J 


=1     fl    •- 


£  a  -^ 


eo   o 


LO    >0  O  ■*    •<#  (M  TtH 

eo  t^  00  (N  o  CO  c<j 

00   -*  (M  t^   (M  CO  T-H 

o  o  o  o  o  o  o 

i-i  (m'  CO   ■*"  iC 


lO 


.a-s  >, 


I    QO<roooo5cot>.iOi-i 

COOlNi— i-*cO00»Oi-H 
i-(T-iOO(MTfliCt>.C<l 
OCOOOOOOOO 


•-   TtH    CO     M    ^ 

(U  ^  ^  ^  — . 


,nJ   o   O 


.2i.  o 


a  a 


;:j     CO     z:     en     rj     en 

s  g  ff  a  a  g 

bO  .O     bo  _0     to  ,0 


a 

bo 

_H 

en 

O 

a 

bO 

iO 

iH 

•c 

CO 

cn- 

CM 

o 

D 

CO 

1 

T! 

D, 

1 

Oi 

o 

00 

00 


S  3 

n3 

fl  tSJ 

3  B 


300  Mary  Davies  Swartz, 

Excluding  the  experiment  of  Luchsinger  (310)  which  was  estimated 
on  a  very  low  specific  rotation  for  glycogen,  only  four  out  of  the  17 
experiments  before  Miura's  (313)  are  positive,  and  in  these  the  gly- 
cogen was  estimated  without  purification,  so  that  the  figures  are  prob- 
ably high.  In  more  reliable  experiments  of  Miura  (313),  and  Mendel 
and  Nakaseko  (312),  the  glycogen  content  of  the  rabbits'  livers  was 
as  low  or  lower  than  the  starvation  maximum  for  the  rabbit,  as  esti- 
mated by  Klilz  (309),  so  that  glycogen  formation  from  inulin  must  be 
regarded  as  doubtful,  or  very  slight. 

When  inuhn  is  introduced  parenterally  into  the  organism,  there  is 
no  inversion  or  utilization,  as  shown  by  the  experiments  of  Mendel 
and  Mitchell  (311).  They  injected  warm  solutions  into  the  peritoneal 
cavity,  and  determining  the  output  of  inulin  in  the  urine  (which  was 
sugar-free)  by  calculations  from  the  specific  rotation,  recovered  2.2 
grams  of  2.8  grams  injected.  In  an  experiment  in  which  the  sugar- 
free  urine  was  hydrolyzed,  and  the  output  of  inulin  calculated  from 
the  amount  of  reducing  sugar  obtained,  1.43  grams  were  recovered 
out  of  2.2  grams  injected.  Weinland  (326)  after  subcutaneous  injec- 
tions of  inulin  into  dogs,  continued  for  a  month,  found  no  inulase 
produced  thereby.  On  the  other  hand,  Saiki  (316)  succeeded  in  pro- 
ducing a  definite  anti-inulase  in  rabbit's  serum. 

We  see,  therefore,  that  inuUn  is  not  attacked  by  animal  enzymes,  as 
far  as  investigated,  with  the  possible  exception  of  two  species  of  inver- 
tebrates; and  by  a  very  few  vegetable  enzymes.  It  appears  to  a  con- 
siderable extent  in  the  faeces  after  being  fed  per  os  in  spite  of  the  abil- 
ity of  the  gastric  juice  to  hydrolyze  it.  In  spite  of  the  accepted  fact 
that  levulose  is  capable  of  being  directly  utilized  by  the  animal  body 
there  is  no  conclusive  evidence  of  glycogen  formation  from  inuUn. 
Whether  other  levulans  resemble  this  hemicellulose  in  these  respects 
has  not  been  investigated. 

Occurrence  and  Nature  of  Dextrans. 

In  the  higher  plants,  starch,  dextrin,  and  cellulose  occur  almost  to 
the  exclusion  of  other  anhydrides  of  dextrose.  A  few  hemicelluloses 
yielding  dextrose  have  been  described,  however,  such  as  "a-amylam" 
(soluble  in  hot  water)  and  "|3-amylam"  (soluble  in  cold  water),  dis- 
covered by  0  'Sullivan  (343)  in  wheat,  rye  and  barley;  those  in  the 
mucilaginous  extracts  of  flax-seed  and  fleabane,  described  by  Bauer 
(329)  and  Rothenfusser  (345);  and  that  in  Colocasia  antiquorum, 
described  by  Yoshimure  (352). 


Nutrition  Investigations.  301 

Even  in  the  lower  plants,  dextrans  do  not  occur  to  any  great  extent. 
They  have  been  observed  in  bacteria  (338),  yeast  (339),  fungi  (350), 
and  liverworts  (337),  but  occur  most  abundantly  in  Hchens  and  algae^ 
the  lichens,  as  already  stated,  yielding  dextrans  to  which  the  names 
lichenin,  isolichenin,  usnin,  everniin,  etc.,  have  been  given.  Especial 
interest  is  attached  to  the  dextrans  of  Cetraria  islandica  (Hchenin  and 
isoKchenin)  which  together  form  80-90  per  cent  of  the  total  carbohy- 
drates of  this  lichen,  because  of  its  abundance  in  northern  lands  and 
its  use  there  as  a  foodstuff;  hence  these  carbohydrates  have  received 
more  attention  from  chemical  investigators  than  any  other  dextrans. 
Ever  since  Berzelius  (333),  in  1808,  studied  the  hot  water  extract  of 
Cetraria  islandica,  and  called  the  carbohydrate  mixture  so  extracted 
"moss-starch,"  on  account  of  its  giving  a  blue  color  with  iodine,  the 
idea  that  it  is,  like  starch,  a  valuable  nutrient,  has  prevailed.  That 
this  hot  water  extract  contained  two  carbohydrates,  one  soluble  in 
cold  water  (isoUchenin)  and  the  other  in  hot,  was  demonstrated  by 
Berg  (332)  in  1873,  who  also  showed  that  the  blue  coloration  with 
iodine  was  a  property  of  isohchenin,  but  not  of  lichenin.  Lichenin  was 
first  found  to  yield  dextrose  by  Klason,  in  1886  (337).  The  next  year 
the  two  carbohydrates  were  more  fully  investigated  by  Honig  and  St. 
Schubert  (336),  who  have  carefully  reviewed  the  earher  Hterature  on 
this  subject.  That  Hchenin  and  isolichenin  yield  dextrose  on  hydroly- 
sis, has  been  verified  by  Karl  Miiller  (341),  Brown  (334),  and  Ulander 
(348) ,  who  have  also  shown  the  hemicelluloses  of  the  water-insoluble 
part  to  consist  of  dextran,  mannan,  and  galactan,  with  a  small  amount 
of  pentosan.  Escombe's  (335)  observation  that  lichenin  yields  gal- 
actose has  proved  to  be  incorrect. 

DEXTRANASES  IN  THE  VEGETABLE  KINGDOM. 

Honig  and  St.  Schubert  (336)  subjected  isolichenin  to  the  action  of 
malt  diastase,  and  observed  a  rapid  disappearance  of  the  iodine  color 
reaction,  and  the  formation  of  a  dextrin-Hke  substance  precipitable 
by  alcohol  —  a  result  verified  by  Brown  (334)  in  1898.  Berg  (332) 
treated  lichenin  with  malt  diastase  but  was  unable  to  observe  any 
change  produced  in  it;  his  results  also  have  been  verified  by  Brown 
(334).  The  only  experiments  in  which  sugar  has  been  obtained  from 
lichenin  by  the  action  of  vegetable  enzymes  have  been  carried  out  by 
Saiki  (346)  with  "Taka"  diastase  from  Eurotium  oryzae  and  inulase 
from  Aspergillus  niger. 

^Cf.  p.  255,  also  V.  Lippmann,  Chemie  der  Zuckerarten,  Vol.  I,  pp.  215-220. 


302  Mary  Davies  Swartz, 

DEXTRANASES  IN   THE   ANIMAL   KINGDOM. 

Attempts  to  hydrolzye  lichenin  by  animal  enzymes  have  been  uni- 
formly unsuccessful.  The  most  exhaustive  researches  were  made  by 
Nilson  (342),  in  1893,  partly  with  pure  lichenin  and  partly  with  the 
powdered  lichen  itself.  Digestions  were  made  with  human  gastric  juice 
for  24  hours,  in  neutral,  acid,  and  alkaUne  solutions;  with  pancreatic 
extracts;  with  gastric  juice  followed  by  pancreatic  extract;  and  with 
these  same  extracts,  using  preparations  treated  with  \  per  cent  sodium 
hydroxide  solution  for  24  hours  before  the  digestion.  Nilson  signifi- 
cantly remarks  that  this  resistance  to  sugar-forining  enzymes  is  worthy 
of  note,  inasmuch  as  certain  lichens  have  been  considered  valuable  food 
for  man,  and  that  it  is  hard  to  understand  how  reindeer  utilize  the  car- 
bohydrates of  lichens.  His  negative  results  with  animal  enzymes  have 
been  substantiated  by  Brown  (334) — who  found  digestion  with  0.2  per 
cent  to  0.4  per  cent  hydrochloric  acid  equally  ineffective  —  and  by 
Saiki  (346).  Torup  (347)  reports  that  the  dextran  isolated  from  La- 
minaria  digitata  by  Krefting  is  not  hydrolyzed  by  ptyalin,  amylopsin 
or  diastase. 

DIGESTION  AND  UTILIZATION  IN  ANIMALS  AND   MAN. 

Interest  in  the  digestibility  of  lichenin  arises,  not  only  from  its  use 
in  the  diet  of  normal  individuals,  but  in  the  possibihty  of  its  furnish- 
ing a  substitute  for  other  carbohydrates  in  the  diet  of  diabetics. 
After  this  idea  was  set  forth  by  Kiilz  (305),  in  1874,  it  is  not  surprising 
to  find,  in  1879,  the  Italian  physician  Cantani,i  and  the  Norwegian 
physician  Bugge^  reporting  experiments  in  the  use  of  Cetraria  bread 
for  diabetics.  Without  any  further  observations  than  that  the  sugar 
in  the  urine  was  not  increased,  the  idea  prevailed  which  Voit  expres- 
sed in  his  monograph  on  Nutrition  in  1881  (348)  and  Poulsson  repeated 
in  1906  (344),  that  in  some  way  or  other,  the  "moss-starch,"  or 
lichenin,  was  changed  into  sugar  in  the  ahmentary  tract,  and  served  as 
a  true  nutrient.  Poulsson  undertook  to  verify  this  by  feeding  experi- 
ments with  two  diabetics,  but  as  Mendel  (340)  has  taken  pains  to 
point  out,  the  results  obtained,  namely  that  45^9  per  cent  of  the  car- 
bohydrates of  the  Cetraria  bread  eaten  were  utiUzed,  are  unreHable, 
since  the  carbohydrates  of  the  faeces  were  calculated  by  difference, 
instead  of  being  determined  directly  by  analysis. 

iCited  by  Poulsson  (344). 

^Bugge,  Forhandlingar  i  det  medicinske  selskap,  Kristiania,  1879,  p.  179  (cited  by 
Poulsson). 


Nutrition  Investigations. 


303 


The  few  feeding  experiments  made  with  animals  do  not  sustain  the 
claims  made  for  the  value  of  Cetraria  as  a  foodstuff.  Brown  (334) 
found  only  1.25-0.7  per  cent  glycogen  in  the  Uvers  of  rabbits  after 
Cetraria  feeding,  but  these  results  are  not  very  satisfactory,  since  the 
rabbits  would  not  eat  it  very  well.  An  old  experiment  by  von  Mering 
(351),  in  which  16  grams  iichenin  were  fed  to  each  of  two  rabbits, 
shows  0.56-0.63  grams  of  glycogen  in  the  Hver,  but  Miura  (313)  has 
pointed  out  that  his  glycogen  estimates  were  probably  too  high.  Saiki 
(346)  fed  Cetraria  extract,  containing  2  per  cent  dry  matter,  in  por- 
tions of  292  cc.  and  300  cc.  on  two  successive  days,  to  a  meat-fed  dog. 
The  faeces  of  the  feeding  period  were  marked  off  at  the  beginning  of 
the  Cetraria  diet  by  fine  quartz,  and  at  the  end  by  cork.  Their  com- 
position is  s'hown  in  the  following  table: 


DIET 

i 

AS  DEXTROSE. 

Meat 

2  days 
2  days 
2  days 
2  days 

10 
15* 

5* 

6 

5.8 
25.8 
24.5 

3.2 

0.68 

Meat  +  Cetraria  extract . . 
Meat 

3.90 
1.20 

Meat 

0.19 

*  Faeces  of  Cetraria  Period. 

The  Cetraria  extract  contained  6.3  grams  carbohydrate  estimated 
as  dextrose,  the  faeces  5.1  grams. 

Feeding  experiments  on  man,  in  which  the  intake  and  output  of 
carbohydrate  have  been  carefully  determined  by  direct  analysis  of 
the  carbohydrate  as  dextrose,  have  recently  been  conducted  in  Pro- 
fessor Mendel's  laboratory.  The  data  have  not  yet  been  pubHshed  in 
detail,  but  from  a  preliminary  description  given  by  Mendel  (340) 
is  taken  the  following  report  of  one  experiment* : 


I.    Fore  period  =  3  days 

Cetraria  period  =  3  days 

II.    Fore  period  =  2  days 

Fore  period  =  daily 

Cetraria  period  =  1  day 

After  period  =  2  days 

*  From  unpublished  experiments  by  Dr.  V, 
cal  Chemistry. 


TAECES. 

Weight  Air  Dry. 


Grams. 

35 

146 

68 
34 
63 

29 


CARBOHYDRATE. 

As  Dectrose. 


Grams. 
2.1 

38.0 

6 

6 

24 


Per  cent. 

1 

56 

4 

2 

13 


CETRARIA  FED. 


80  g.  =  56g. 
as  dextrose 


20g.  =  14|g. 
as  dextrose 


C.  Meyers,  Sheffield  Laboratory  of  Physiologi- 


304 


Mary  Davits  Swartz, 


In  this  experiment,  the  Cetraria  islandica  was  carefully  washed, 
extracted  with  a  dilute  solution  of  potassium  carbonate,  to  remove 
the  bitter  principle;  again  thoroughly  washed,  dried  and  ground  to 
a  powder.  This  preparation  contained  72.5  per  cent  carbohydrate  as 
dextrose.  The  carbohydrates  of  the  diet,  throughout  the  experiment, 
were  limited  to  fine  white  bread  and  zwieback,  forms  in  which  they  are 
utilized  in  man  to  98  per  cent.  The  faeces  were  hydrolized  with  dilute 
acid,  and  the  carbohydrates  determined  as  dextrose  by  Allihn's  gravi- 
metric method.  It  is  evident  that  nearly  all  of  the  Cetraria  carbo- 
hydrate escaped  digestion  and  was  recovered  in  the  faeces. 

Through  the  kindness  of  Professor  Mendel,  the  protocol  of  a  similar 
experiment,  by  Mr.  S.  W.  MacArthur,  is  also  reproduced,  in  which 
the  technique  was  practically  the  same  as  described  for  Dr.  Myers's 
experiment. 


DIET. 

COMPOSITION   OP  THE  TAECES.      ' 

Cellulose-Free. 

Weight 
Moist. 

Weight 
Air  Dried. 

Dextrose. 

Dextrose. 

Fore   =  3  days ....    Meat,  etc. 

Mid    =  3  days ....    Meat  +  Cetraria* 

After  =  3  days .  .  .  . '  Meat,  etc. 

Grams.          Grams. 

281           90.0 
542         149.0 

284      !    87.5 

Per  cent. 

4.4 

27.6 

4.9 

Grams. 

3.96 

34.5* 

4.2 

*  Amount  Cetraria  eaten  =  47  grams,  which  would  be  equivalent  to   34.1  grams   of  dextrose 
in  faeces. 


It  is  evident  that  the  results  of  this  experiment  simply  confirm  those 
of  Dr.  Myers,  and  demonstrate  that  uncooked  Cetraria,  although 
taken  in  a  form  as  favorable  as  possible  for  its  digestion,  is  scarcely 
affected  by  its  passage  through  the  alimentary  canal,  and  must  be 
classed  among  the  indigestible  carbohydrates.  Very  desirable  expe- 
riments on  the  digestibility  of  the  peculiar  carbohydrate  of  Cetraria  — 
lichenin  —  are  also  being  conducted,  which  may  throw  new  light  on 
the  digestibility  of  the  dextrans,  but  at  present  we  certainly  have  no 
grounds  for  assuming  that  this  group  of  hemicelluloses  deserves  to  be 
classed  with  the  true  nutrients;  all  experiments  show  that  they  are 
not  attacked  by  animal  enzymes,  and  are  recovered  unchanged  in  the 
faeces  after  feeding. 

In  conclusion,  attention  may  be  called  to  certain  data  from  Japan- 
ese dietary  studies,  given  by  Oshima  (15),  as  to  the  digestibility  of 


Nutrition  Investigations. 


305 


some  dried  marine  algae,  which  have  not  been  mentioned  in  connec- 
tion with  the  different  classes  of  hemic elluloses.  The  coefficient  of 
digestibility  for  each  species  studied  is  given  in  the  following  table: 


ALGAE    DRIED. 

OTHER    SUBSTANCES    IN    DIET. 

COEFFICIENT    OF 
DIGESTIBILITY 

(Carbohydrates  includ- 
ing crude  fiber). 

Ecklonia  bicyclis 

Shoyu*  and  sugar 

Shoyu 

Shoyu  and  cleaned  rice 

Cleaned  rice,  shoyu,  sugar 

36.2 

75.2 

Laminaria  sp  .       

55.0 

Ulopteryx  pinnatifida 

72.3 

Average . 


67.7 


*  Soy-bean  sauce. 


III.    EXPERIMENTAL  PART. 

Introduction. 

The  foregoing  review  has  emphasized  the  limits  of  our  knowledge, 
both  in  regard  to  the  chemical  composition  of  marine  algae,  and  their 
fate  in  the  alimentary  tract  of  men  and  animals,  as  determined  by 
actual  measurement  of  intake  and  output,  and  as  explained  by  the  ac- 
tion of  bacteria  and  enzymes  in  vitro.  Ten  species  of  marine  algae 
have,  therefore,  been  made  the  basis  of  the  present  investigations. 
Eight  of  them  were  Hawaiian  Limu,  obtained,  as  already  stated, 
through  the  kindness  of  Miss  Minnie  Reed,  Science  teacher  in  the  Ka- 
mehameha  Boys'  School,  Honolulu.  They  were  dried  in  the  sun,  with 
the  salt  water  adhering  to  them,  before  shipping  to  America.  The 
other  two  (dulse  and  Irish  moss)  were  easily  obtained  in  our  Eastern 
markets. 

That  the  carbohydrates  of  algae  are  chiefly  hemicelluloses,  is  indi- 
cated by  the  analyses  which  have  already  been  made;  that  in  many 
species,  these  are  to  a  great  extent  water-soluble,  is  also  well  known. 
In  as  much  as  such  soluble  forms  are  thus  particularly  well  adapted 
for  nutrition  investigation  on  account  of  their  freedom  from  all  in- 
crusting  substances,  which  end  to  interfere  with  digestion,  the  present 
studies  have  been  confined  as  far  as  possible  to  them.  Since  it  was 
desirable  to  study  the  different  groups  of  hemicelluloses,  and  man- 
nans  and  levulans  were  not  found  in  the  seaweeds  in  sufi&cient  quanti- 
ties for  metaboHsm  experiments,  these  were  obtained  from  other 
sources;  a  mannan  from  salep,  and  a  levulan  (sinistrin)  from  squills 
(S cilia  maritima). 

Other  investigators  in  this  laboratory  are  working  on  a  dextran 
which  would  naturally  be  included  here,  namely  lichenin  from  Cetra- 
ria  islandica;  consequently  no  experimental  studies  on  this  group  of 
hemicelluloses  have  been  made.  In  considering  any  classifications 
of  these  materials,  it  must  be  borne  in  mind  that  most  of  these  carbo- 
hydrates are  more  or  less  complex  in  nature,  and  can  be  grouped  only 
with  reference  to  what  appears  to  be  the  chief  constituent  in  any 
given  case.  The  following  list  comprises  al  the  species  examined, 
arranged  upon  this  plan: 

306 


Nutrition  Investigations.  •    307 

I.  The  Pentosans: 

Dulse  {Rhydomenia  palmata), 

Limu  Lipoa  {Haliseris  pardalis), 

Limu  Eleele  {Enter omorpha  intestinalis) , 

Limu  Pahapaha  {Ulva  lactuca  laciniata  and  Ulvafasciata).     ■■ 

II.  The   Galactans: 

Irish  Moss  {Chondrus  crispus), 

Limu  Manauea  {Gracilaria  coronopifolia), 

Limu  Huna  {Eypnea  nidifica), 

Limu  Akiaki  {Ahnfeldtia  concinna), 

Limu  Uaualoli  {Gymnogongrus  vermicularis  americana  and 

Gymnogongrus  disciplinalis) , 
Limu  Kohu  {Asparagospis  sanfordiana) , 
Slippery  Elm  (JJlmus  Juha) . 

III.  The   Mannans: 

Salep  (Species  of  Orchis  and  Eulophia). 

IV.  The  Levulans: 

Sinistrin  {Urginea  or  S cilia  maritima). 

The  primary  object  of  these  investigations  has  been  to  determine 
the  fate  of  these  substances  in  the  alimentary  canal  of  man,  since  they 
are  all  used  as  foodstuffs  except  sinistrin,  and  are  all  representative 
of  a  large  class  of  materials  so  employed.  The  experiments  con- 
ducted have  been  Chemical,  Bacteriological  and  Physiological 
in  character,  and  each  of  these  phases  will  be' taken  up  separately  in 
turn  in  the  following  pages. 

Chemical  Investigations. 

The  aim  of  the  experiments  was  to  isolate,  identify,  and  pre- 
pare for  bacteriological  and  physiological  experiments,  any  water- 
soluble  carbohydrates  present  in  sufficient  amoimt  in  the  materials 
under  consideration;  and  to  determine  such  of  their  properties  as 
would  facilitate  their  detection,  isolation,  and  quantitative  estimation 
in  these  experiments. 

general  methods. 

All  the  seaweeds,  with  the  exception  of  Irish  moss,  were  washed  re- 
peatedly in  cold  tap  water,  to  remove  salt,  sand,  and  other  foreign 
substances,  and  for  convenience,  dried  by  spreading  in  thm  layers 


308  Mary  Davies  Swartz, 

over  steam  radiators.  The  Irish  moss,  being  comparatively  free  from 
salt,  etc.;  and  largely  soluble  in  pure  water,  was  quickly  washed  once, 
and  extracted  immediately. 

All  hydrolyses  of  carbohydiatts  were  made  with  2  per  cent  hydro- 
chloric acid,  by  boiling  with  a  reflux  condenser  over  a  free  flame. 
After  coohng,  the  acid  was  neutral' zd  with  potassium  hydroxide, 
using  phenolphthalein  as  an  indicator,  when  the  solutions  were  suf- 
ficiently Hght  in  color;  in  other  cases,  litmus  paper  was  employed. 
When  the  products  of  hydrolysis  served  to  determine  the  nature  of 
the  carbohydrates,  they  were  evaporated  on  a  water  bath  nearly  to 
dryness,  the  residues  extracted  with  hot  95  per  cent  alcohol  the  alcohol 
removed  from  the  filtered  solution  by  evaporation,  the  residues  fre- 
quently taken  up  in  a  little  water  and  decolorized  with  charcoal,  con- 
centrated, and  again  extracted  with  absolute  alcohol. 

All  qualitative  tests  for  reducing  sugar  were  made  with  Fehling's 
solution;  all  quantitative  tests  by  AUihn's  gravimetric  method  for 
dextrose,  the  results  being  calculated  as  dextrose  in  view  of  the  com- 
plex nature  of  most  of  the  products,  and  the  advantage  of  uniformity. 
On  all  preparations  used  for  feeding  experiments,  the  length  of  time 
in  which  the  maximum  yield  of  sugar  could  be  obtained  has  been  de- 
termined, as  a  criterion  in  analyses  of  faeces.  Five  grams  of  dry  air 
material  were  hydrolyzed  in  500  cc.  of  2  per  cent  hydrochloric  acid, 
50  cc.  being  removed  at  intervals  of  one  or  more  hours,  cooled,  neu- 
tralized, made  up  to  100  cc.  and  reducing  power  determined  as  dex- 
trose by  Allihn's  gravimetric  method. 

Tests  for  the  presence  of  fermenting  sugars  have  been  made  in 
fermentation  tubes  with  fresh  compressed  yeast,  using  as  controls 
solutions  of  the  substance  to  be  tested,  without  yeast,  and  dextrose 
solutions  with  yeast. 

All  carbohydrate  solutions  for  polariscopic  examination  have  been 
clarified  by  addition  of  an  equal  volume  of  alumina  cream. 

Qualitative  tests  for  pentosans  have  been  made  by  boiling  the  sub- 
stance to  be  tested  in  a  small  Erlenmyer  flask  with  12  per  cent  hydro- 
chloric acid  and  testing  for  furfurol  with  anihn-acetate  paper. 

Quantitative  tests  for  pentosans  have  been  made  by  the  furfurol- 
phloroglucin  method.^ 

Tests  for  galactans  or  galactose  have  been  made  by  oxidation  with 
nitric  acid  to  mucic  acid,  and  the  mucic  acid  identified  by  Its  melting 
point  (212°  C.-215°  C). 

^Described  in  "Official  and  Provisional  Methods  of  Analysis,"  United  States 
Department  of  Agriculture,  Bureau  of  Chemistry,  Bull.  No.  107,  1907. 


Nutrition  Investigations.  309 

Qualitative  tests  for  mannose  have  been  made  by  Storer's  (271) 
method.  The  products  of  hydrolysis,  freed  from  the  greater  part  of 
the  salts,  gums,  etc.,  in  the  manner  already  described,  were  taken 
up  in  a  Httle  water,  and  portions  of  1  cc.  or  2  cc.  placed  in  test 
tubes.  The  reagent  for  testing  was  freshly  prepared  by  shaking 
together  1  cc.  of  phenylhydrazin,  2  cc.  of  glacial  acetic  acid,  and 
10  cc.  of  distilled  water.  3-16  drops  of  this  reagent  were  added  to 
each  of  the  test  tubes,  and  after  standing  several  hours  at  room  tem- 
perature, they  were  examined  for  precipitates  of  mannose-hydrazone. 
These  precipitates  were  examined  under  the  microscope,  because  they 
usually  contained  considerable  amorphous  matter.  The  mannose- 
hydrazone  itself  does  not  come  down  as  colorless  rhombic  plates  at 
first,  but  as  globules  of  greenish-yellow  or  brownish-yellow  color, 
sometimes  smooth  and  resembhng  large  yeast  ceUs  in  the  way  they 
c  uster  together,  and  at  other  times  covered  with  blimt  points  or 
spines.  When  these  globules  were  observed,  the  precipitate  was  care- 
fully washed  with  water,  sometimes  without  removing  from  the  test- 
tube,  the  last  drops  being  taken  up  with  filter  paper,  and  then  dissolved 
in  warm  diluted  alcohol  (3  parts  of  95  per  cent  to  1  part  water),  which 
was  not  filtered,  but  decanted  from  the  amorphous  insoluble  portion, 
and  allowed  to  evaporate  slowly  to  facihtate  the  formation  of  crys- 
tals. Unless  these  crystals  could  be  obtained,  the  tests  were  consid- 
ered negative,  although  Storer  has  pointed  out  that  they  are  sometimes 
difi6cult  to  obtain,  even  when  true  mannose-hydrazone  balls  are 
present. 

All  quantitative  determinations  have  been  made  in  duplicate  un- 
less otherwise  stated. 

PENTOSAN  PREPARATIONS. 

Dulse. 

A  pure,  water-soluble  pentosan-preparation  has  been  obtained  from 
dulse  (Rhodymenia  palmata).  After  boihng  in  water,  in  an  open 
vessel,  with  occasional  stirring,  for  several  hours,  this  dark,  reddish- 
brown  seaweed  yielded  a  carbohydrate,  non-mucilaginous  in  character, 
which  could  be  precipitated  from  its  solutions  by  alcohol.  About  12 
hours'  boiling  proved  to  be  necessary  for  complete  extractions.  The 
hot,  brown,  watery  extract  was  first  filtered  through  gauze,  and  then 
through  cotton,  as  it  clogged  up  filter  paper  very  quickly.  This 
filtrate,  concentrated  to  a  syrup  on  a  water  bath,  was  poured  while 


310  Mary  Davies  Swartz, 

still  warm  into  about  three  times  its  volume  of  acetone,  which  expe" 
rience  showed  to  be  a  more  satisfactory  precipitant  than  alcohol- 
Most  of  the  carbohydrate  came  down  very  soon,  in  large,  flocculent^ 
yellowish-white  masses,  but  a  portion  remained  in  suspension  as  a 
fine  white  powder,  which  made  filtration  difficult.  The  bulk  of  the 
precipitate  was  therefore  removed  by  filtering  through  three  or  four 
thicknesses  of  fine  gauze,  and  the  rest  obtained  by  distilling  oS  the  ace- 
tone, concentrating  the  residue,  and  reprecipitating  the  carbohydrate 
in  solution  with  acetone.  This  precipitate  was  very  hydroscopic,  and 
was  therefore  transferred  immediately  to  95  per  cent  alcohol.  This 
was  replaced  by  fresh  alcohol  after  a  few  hours,  and  the  whole  boiled 
on  a  reflex  condenser  for  half  an  hour.  A  yellowish,  granular  powder 
was  thus  obtained,  which  was  filtered,  washed  with  ether,  and  the  ad- 
herent ether  allowed  to  evaporate.  It  was  then  redissolved  in  a  small 
volume  of  water,  filtered  hot  through  paper,  on  a  jacketed  funnel^ 
reprecipitated  with  acetone,  again  put  into  95  per  cent  alcohol,  and 
finally  into  absolute  alcohol,  in  which  it  was  allowed  to  stand  for 
several  weeks.  It  was  then  filtered  off,  washed  with  ether,  and  dried  in- 
vacuo  over  sulphuric  acid.  The  product  was  a  cream-white  powder, 
and  apparently  not  at  all  hydroscopic.  From  about  two  kilograms  of 
crude  commercial  dulse,  approximately  75  grams  of  this  material  were 
obtained,  and  used  subsequently  for  feeding  experiments. 
'':Jin  attempt  made  to  remove  the  dark  red  coloring  matter  by  extrac- 
tion with  1  per  cent  sodium  carbonate,  led  to  the  discovery  that  this 
carbohydrate  is  readily  extracted  by  dilute  alkaline  solutions.  For 
preparations  on  a  large  scale,  it  was  therefore  found  more  satisfactory 
to  use  the  following  method,  based  on  Salkowski's  method  (139,  140) 
of  obtaining  xylan  and  araban  by  precipitation  with  Fehling's  solu- 
tion. This  method  could  be  applied  exactly  as  described,  but  there 
was  an  evident  tendency  for  the  carbohydrate  to  dissolve  in  the  Feh- 
ling's solution. 

The  dulse  was  accordingly  extracted  with  1  per  cent  potassium 
hydroxide  solution  for  48  hours,  with  occasional  stirring,  the  extract 
removed  by  a  hand  press,  and  the  extraction  with  fresh  alkali  repeated 
for  24  hours.i  These  extracts  were  filtered  through  several  thicknesses 
of  gauze,  and  to  this  filtrate  a  solution  of  copper  sulphate  was  added 
till  the  reaction  was  just  neutral.  A  flocculent,  bluish-green  precipi- 
tate formed.  Into  this  solution  was  stirred  carefully  the  alkaline 
Rochelle  salt-potassium  hydroxide  solution  used  for  Fehling's  solu- 
tion, until  the  precipitate  clumped  together  in  heavy  granular  masses. 

^A  third  extraction  contained  so  little  of  the  material  that  it  was  discarded. 


Nutrition  Investigations.  311 

This  was  easily  filtered  off  through  gauze,  as  much  liquid  as  possible 
removed  by  pressure,  and  the  precipitate  washed  quickly  with  a 
little  water  to  remove  the  excess  of  alkali.  The  carbohydrate  was 
freed  from  its  copper  compound  just  as  described  by  Salkowski  (140). 
The  precipitate  was  placed  in  a  mortar  and  rubbed  to  a  cream  with 
diluted  hydrochloric  acid  (1  volume  of  water  to  1  volume  of  concen- 
trated acid)  the  acid  being  added  \mtil  aU  blue  particles  had  disap- 
peared. It  was  then  poured  into  90  per  cent  alcohol,  the  precipitate 
filtered  off  upon  plaited  paper  and  washed  with  50  per  cent  alcohol, 
replaced  in  90  per  cent  alcohol  acidified  with  hydrochloric  acid,  and 
allowed  to  stand  several  hours  to  dissolve  out  the  copper.  It  was  then 
filtered,  dissolved  in  dilute  potassium  hydroxide,  and  the  dark  brown, 
muddy  solution  filtered  through  paper  on  a  hot  funnel,  the  carbohy- 
drate reprecipitated  with  acid  alcohol,  and  redissolved  and  reprecipi- 
tated  until  free  from  copper.  When  it  no  longer  came  down  readily 
in  alcohol,  acetone  was  substituted,  in  which  it  formed  white  fibrous 
masses  resembKng  paper  pulp.  Washed  with  absolute  alcohol  and 
ether,  and  dried  in  vacuo  over  sulphuric  acid,  it  became  a  cream-white 
powder.  Both  of  these  methods  yielded  a  product  readily  soluble  in 
cold  water,  forming  a  clear,  limpid,  amber-colored  solution.  It  gave 
no  color  reaction  with  iodine,  and  contained  no  reducing  substance. 
In  Fehling's  solution  it  formed  a  very  flocculent  white  precipitate,  was 
not  precipitable  by  lead  acetate,  neutral  or  basic,  in  neutral  solution, 
but  formed  a  precipitate  in  alkaline  solutions.  A  test  for  mucic  acid 
gave  negative  results,  but  a  strong  furfurol  reaction  was  obtained  on 
boiling  with  hydrochloric  acid,  indicating  the  presence  of  pentosans. 
A  1-gram  sample  of  material,  prepared  by  the  method  first  described, 
was  tested  quantitatively  for  pentosans.  It  contained  26.8  per  cent 
moisture,  and  2.48  per  cent  ash,  and  yielded  0.076  grams  of  phloro- 
glucid,  from  which  the  yield  of  pentosans,  according  to  Krober's 
tables,!  is  calculated  as  72  per  cent.  The  phoroglucid  precipitates  were 
afterwards  extracted  with  95  per  cent  alcohol,  according  to  EUett 
and  ToUen's^  method  for  quantitative  determination  of  methyl-fur- 
furol.  The  Gooch  crucibles  containing  the  precipitates  were  warmed 
10  minutes  to  60°  C.  with  15-20  cc.  of  alcohol,  the  extract  filtered  off, 
and  the  extraction  repeated  till  the  alcohol  was  colorless.  The  pre- 
cipitates were  then  dried  at  100°  C.  and  weighed.  The  loss  of  weight 
was  0.0047  grams  or  6  per  cent  of  the  original  precipitate.  The  dulse 
preparation  therefore  contained  a  small  amount  of  methyl-pentosan 

^Zeitschrift  fiir  physiologische  Chemie,  XXXVI,  appendix, 

^Berichte  der  deutschen  chemischen  Gesellschaft,  Vol.  38,  p.  492  (1905). 


312  Mary  Davies  Swartz, 

The  products  of  hydrolysis  were  tested  for  fermenting  sugar,  with 
negative  results,  but  after  heating  with  phenyl-hydrazin-hydrochloride 
and  sodium  acetate,  an  abundant  yield  of  osazones  was  obtained. 
These  cr>^stallized  out  only  on  cooling,  were  pale  yellow,  soluble  in 
hot  water  only  with  great  difficulty,  but  very  soluble  in  alcohol, 
acetone,  or  pyridin.  After  four  or  five  recrystallizations  from  alcohol, 
they  melted  at  152°  C.  and  this  melting  point  remained  constant  after 
ten  or  twelve  recrystallizations.  However,  there  were  very  minute 
points  at  which  melting  seemed  to  occur  about  140°  C.  Under  the 
microscope,  clusters  of  long  needles  were  seen,  each  with  a  tuft  of 
small  fine  needles  springing  from  its  very  tip.  Dissolved  in  glacial 
acetic  acid,  and  examined  in  a  100  mm.  tube,  these  osazones  showed 
no  rotation  of  polarized  light. 

A  very  white  sample  of  the  dulse  carbohydrate  was  used  to  deter- 
mine its  specific  rotation.  It  contained  7.1  per  cent  moisture  and 
1.68  per  cent  ash.  Two  determinations  were  made,  one  on  a  0.6  per 
cent  solution  and  the  other  on  a  1.0  per  cent  solution  for  which  the 
polariscope  readings  in  a  200  mm.  tube  were  respectively  —0.90° 
and  —1.52°.  The  specific  rotation,  calculated  from  these  readings 
was  therefore  [aj^  =  —75.2°  and  —76.2°,  or  corrected  for  moisture 
and  ash,  [a]^  =   -82.4°  and  -83.6°,  average,  -83°. 

The  rate  of  hydrolysis  and  maximum  reducing  power  were  deter- 
mined as  follows:  5  grams  of  the  material  dissolved  in  500  cc.  of  2 
per  cent  hydrochloric  acid  wxre  boiled  in  the  usual  way.  At  the  end 
of  two  hours,  and  at  intervals  of  one  hour  thereafter,  50  cc.  portions 
were  removed,  neutralized  and  made  up  to  100  cc,  and  the  amount 
of  reducing  sugar  present  determined  as  dextrose.  The  following 
results  were  obtained: 

TIME   OF   BOILING.  SUGAR   AS   DEXTROSE. 

Hours.  Per  cent. 

2  87.2 

3  87.2 

4  89.4 

5  89.5 

That  the  results  vary  greatly  with  the  concentration,  is  shown  by 
the  fact  that  a  0.3  per  cent  solution  boiled  5  hours  yielded  67.1  per 
cent  of  sugar  as  dextrose. 

Having  established  the  fact  that  this  dulse  preparation  consists  of 
pentosans,  with  the  properties  described,  further  investigations  into 
the  exact  chemical  nature  of  the  carbohydrates  composing  it  were 
not  considered  within  the  province  of  this  work. 


Nutrition  Investigations.  313 

Hawaiian  Seaweeds. 

Beside  the  dulse  preparation,  three  seaweeds  have  been  included  ia 
this  group  which  yielded  little  or  no  soluble  carbohydrates,  namely, 
Limu  Lipoa  {Haliseris  pardalis),  Limu  Eleele  {Enteromorpha  intesti- 
nalis)  and  Limu  Pahapaha  {Uha  lactuca,  etc.). 

Limu  Lipoa.  Limu  Lipoa  contained  a  small  amount  of  non-muci- 
laginous carbohydrate,  soluble  in  cold  water  as  well  as  hot.  It  was 
precipitated  by  alcohol,  in  which  it  came  down  as  a  white  fibrous 
mass.  On  hydrolysis,  it  yielded  a  dextro-rotatory  fermenting  sugar; 
a  test  with  phenylhydrazin  acetate  for  mannose  was  negative,  as  were 
tests  for  pentosans.  The  total  amount  of  this  carbohydrate  was  so 
small  as  to  be  almost  neghgible,  as  far  as  feeding  experiments  were 
concerned,  hence  the  original  washed  material  was  used,  after  grinding 
to  a  powder  in  a  coffee  mill.  It  contained  a  very  high  percentage  of 
inorganic  matter  because  the  thalU  were  so  encrusted  with  calcareous 
substances,  that  it  was  impossible  to  remove  them  entirely  by  washing. 
This  preparation  gave  a  strong  furfurol  test,  and  a  single  quantitative 
test  for  pentosans  gave  the  following  results: 

The  sample,  weighing  1  gram,  contained  10.5  per  cent  moisture  and 
18.5  per  cent  ash.  It  yielded  0.161  grams  of  phloroglucid,  which 
according  to  Krober's  tables  ^  is  equivalent  to  0.147  grams  pentosans, 
or  14.7  per  cent  of  the  crude  substance. 

Tests  for  starch  and  reducing  sugar  were  negative.  Only  a  minute 
quantity  of  mucic  acid  was  obtained;  a  quantity  too  small  to  purify 
and  determine  the  melting  point.  The  products  of  hydrolysis  showed 
slight  fermentation,  which  was  doubtless  due  to  the  mannan  of  the 
water-extract. 

A  determination  of  the  reducing  power  made  in  the  same  manner 
as  already  described,  gave  the  results: 

TIME  OF  BOILING.  SUGAR  AS  DEXTROSE. 

Hours.  Per  cent. 

11  Very  little 

3  14.3 

4  ■  14.7 
6  12.9 
8  12.8 

Limu  Eleele.  Limu  Eleele  yielded  no  appreciable  amount  of  water- 
soluble  carbohydrate,  even  after  boiling  3  or  4  hours.     The  dried 


1  Zeitschrift  fiir  physiologische  Chemie,  XXXVI,  appendix. 


SUGAR    AS    DEXTROSE. 

Per  cent. 

16.8 

16.9 

18.1 

16.8 

314  Mary  Davie s  Swartz, 

seaweed  was  therefore  simply  finely  ground  for  use  in  feeding  experi- 
ments. 

It  gave  a  strong  furfurol  test,  but  yielded  a  mere  trace  of  mucic 
acid.  Tests  for  starch  and  reducing  sugar  were  negative.  The 
products  of  hydrolysis  contained  no  fermenting  sugar.  From  this  it 
was  e\ddent  that  the  hemicelluloses  were  chiefly  pentosans. 

Determination  of  the  reducing  power  gave  the  following  results: 

TIME   OF   BOILING. 

Hours. 
2 
3 
4 
5 

Limu  Pahapaha.  Ulva  lactuca  is  said  by  Rohmann  (134)  to  con- 
tain a  water-soluble  methyl -pentosan,  rhamnosan;  but  if  this  occurs 
in  Limu  Pahapaha,  it  must  be  in  very  small  amount,  as  an  extract 
of  50  grams  of  the  dried  seaweed,  made  by  boiling  3  or  4  hours,  gave 
very  little  residue  on  evaporation  to  dryness.  For  feeding  experi- 
ments, the  dry  crude  substance  was  simply  ground  to  a  powder. 
Like  Limu  Eleele,  it  gave  a  strong  furfurol  test,  but  yielded  no  mucic 
acid.  Starch  was  present,  but  no  reducing  sugar.  Fermentation 
with  yeast  was  marked  in  12  hours,  probably  due  chiefly  to  the  hy- 
drolysis of  the  starch. 

Determination  of  reducing  power  gave  the  following  results: 

TIME   OF   BOILING.  SUGAR  AS   DEXTROSE. 

Hours.  Per  cent. 

2  28.8 

4  31.8 

GALACTAN    PREPARATIONS. 

Irish  Moss. 

The  carbohydrates  of  Irish  moss  are,  as  already  noted,  readily 
soluble  in  cold  water,  after  the  salt  has  been  removed  from  the  sea- 
weed. By  allowing  the  moss  to  stand  for  24  hours  in  cold  water  (about 
10  liters  to  250  grams  of  dry  substance),  an  almost  colorless,  semi- 
transparent,  mucilaginous  extract  was  obtained.  By  straining  this 
off  through  gauze,  and  allowing  it  to  stand  over  night,  for  minute 
particles  of  cellulose  held  in  suspension  to  settle,  a  solution  almost 
entirely  free  from  insoluble  material  was  obtained  by  decantation. 


Nutrition  Investigations.  315 

This  was  considered  sufficiently  pure  for  feeding  experiments,  and  was 
quickly  dried  by  pouring  into  broad  shallow  dishes  and  placing  over  a 
steam  radiator.  It  formed  yellowish,  translucent  scales,  which  were 
easily  removed,  and  finely  groimd. 

Subsequent  extractions  were  made  in  a  steam  steriHzer,  heating 
several  hours  at  a  time.  Tests  showed  that  the  carbohydrate  was 
not  hydrolyzed  by  this  repeated  subjection  to  high  temperature. 
The  several  extracts  were  first  strained  off  through  gauze  and 
then  filtered  hot  through  cotton,  to  remove  the  cellulose 
particles.  As  these  clogged  even  cotton  filters  very  rapidly,  it 
was  found  most  satisfactory  to  let  the  extracts  stand  over  night, 
decant  off  the  supernatant  fluid  as  far  as  possible,  and  filter 
in  a  water- jacketed  ftmnel.  Solutions  containing  over  1  per  cent 
dry  substance  could  not  be  filtered  through  paper.  For  experi- 
ments where  a  perfectly  clear  fluid  was  desired,  a  |  per  cent 
solution  was  filtered  hot  through  plaited  paper,  and  then  concentrated 
on  a  water  bath  to  the  desired  strength.  One  per  cent  solutions 
formed  a  soft  jeUy  on  cooling;  2  per  cent  solutions,  a  firm  jelly. 

Even  when  evaporated  to  a  thick  syrup,  the  carbohydrates  of  the 
Irish  moss  extract  are  not  readily  precipitated  by  comparatively  large 
volumes  of  95  per  cent  alcohol,  but  form  a  voluminous,  transparent, 
gelatinous  mass.  This  was  found  to  be  more  or  less  characteristic  of 
all  the  galactans  examined.  They  could  be  brought  down  most 
satisfactorily  by  addition  of  sodium  chloride  to  the  extract  before 
pouring  it  into  the  alcohol.  In  this  way  a  white  precipitate  of  fine 
fibers  was  obtained  from  the  moss.  The  carbohydrate  could  also 
be  precipitated  by  saturation  with  potassium  acetate,  and  freed  from 
inorganic  salts  by  dialysis,  according  to  the  method  described  by 
Pohl  (263).  It  could  not  be  precipitated  by  Fehling's  solution,  nor 
by  lead  acetate  in  neutral  solution. 

Owing  to  the  opacity  of  its  solutions,  and  to  the  fact  that  its  gelat- 
inizing property  made  the  use  of  very  dilute  solutions  necessary,  no 
satisfactory  determination  of  its  specific  rotation  could  be  obtained. 
A  0.5  per  cent  solution,  clarified  with  alumina  cream,  and  examined  in 
a  200  nun.  tube,  showed  a  rotation  of  +0.34°,  and  other  trials  gave 
positive  evidence  that  it  was  dextro-rotatory.  The  products  of  hydro- 
lysis were  also  dextro-rotatory,  and  yielded  osazones,  which  after  one 
recrystalHzation  from  alcohol,  had  a  melting  point  of  184°-185°  C. 

The  carbohydrate  gave  a  red- violet  color  with  iodine,  and  con- 
tained no  reducing  sugar.  A  faint  furfurol  test  was  obtained.  Oxi- 
dation with  nitric  acid  gave  a  rich  yield  of  mucic  acid.     Since  Hadike, 


316  Mary  Davies  Swartz, 

Bauer  and  ToUens  (185),  and  Miither  (200)  have  already  shown  that 
Irish  moss  contains  galactan,  levulan,  dextran  and  pentosan  groups, 
these  tests  were  simply  verifications  of  some  of  their  observations. 
Determination  of  the  reducing  power  gave  the  following  results: 


TIME     OF    BOILING. 

SUGAR    AS    DEXTROSE. 

Hours. 

Per  cent. 

2 

45.6 

3 

48.6 

4 

45.8 

Hawaiian  Seaweeds. 

Limu  Manauea  {Gracilaria  cor onopij alia) , 
LimuHuna  (Hypnea  nidifica), 
Limu  Akiaki  {Ahnfeldtia  concinna), 
Limu  Kohu  {Asparagopsis  sanjordiana), 
Limu  Uaualoli  {Gymnogongrus). 

These  five  seaweeds  all  contained  soluble  carbohydrates,  which  were 
extracted  by  boiling  in  water  in  an  open  vessel  over  a  free  flame  for 
two  hours  or  longer,  Limu  Manauea,  Limu  Huna,  and  Limu  Akiaki, 
which  consist  largely  of  soluble  gelatinizing  hemicelluloses,  yielded 
most  of  these  on  boiling  two  or  three  hours.  The  extracts  were  strained 
off  through  gauze,  filtered  hot  through  cotton,  and  dried  in  thin 
sheets  as  described  for  Irish  moss.  While  the  preparations  were  dark 
colored,  and  had  a  decided  "sea"  flavor,  they  were  not  unpleasant, 
and  were  used  in  feeding  experiments  without  further  purification. 
As  already  stated,  the  carbohydrates  were  not  easily  precipitated  with 
alcohol  unless  a  neutral  salt  (as  sodium  chloride)  was  present. 

Limu  Kohu  and  Limu  Uaualoli  contained  only  a  small  proportion 
of  soluble  hemicelluloses,  and  this  was  obtained  only  after  boiling 
8  to  24  hours.  The  extracts  were  also  much  less  gelatinous  in  charac- 
ter. The  thalli  of  Limu  Kohu  are  almost  like  wire  when  dry,  and 
remain  tough  and  hard  even  after  many  hours'  boiling.  The  extracts 
of  these  two  species  were  more  readily  precipitated  by  alcohol  than 
the  others,  but  the  precipitation  was  greatly  facilitated  by  adding 
sodium  chloride.  The  carbohydrate  of  Limu  Kohu  was  precipitated 
as  a  white  cheese-Uke  cake,  floating  on  the  surface,  while  that  of  Uaua- 
loH  came  down  as  a  mass  of  coarse  white  fibers.  These  precipitates 
were  transferred  to  absolute  alcohol,  and  after  standmg  several  days, 
were  filtered  off,  washed  with  ether  and  dried  at  40°-50°  C.    The 


Nutrition  Investigations. 


317 


Kohu  preparation  should  have  been  dried  in  vacuo,  for  it  proved  to 
be  sHghtly  hydroscopic,  and  instead  of  remaining  a  fine  white  powder, 
became  somewhat  brownish.  The  Uaualoli  preparation  dried  easily 
to  a  grayish  white,  Ught,  fibrous  mass. 

Tests  for  starch  and  reducing  sugar  were  negative  on  all  these 
substances.  Tests  for  galactans  and  pentosans  were  positive  in  every 
case.  Three-gram  samples  of  the  air-dry  preparations  of  Limu 
Akiaki,  Limu  Uaualoli  and  Limu  Kohu  respectively  yielded  0.53 
grams,  0.92  grams  and  0.64  grams  of  mucic  acid,  recrystallized  once 
from  ammonium  carbonate.^  The  products  of  hydrolysis  in  no  case 
contained  fermenting  sugars.  It  is  evident  therefore,  that  these  five 
preparations  from  the  foregoing  Hawaiian  seaweeds  consisted  chiefly 
of  galactans,  accompanied  by  some  pentosan-groups.  From  the 
frequency  with  which  methyl-pentosans  have  been  shown  to  occur 
in  all  seaweeds  previously  investigated,  it  is  very  likely  that  they 
occur  in  all  these  varieties  and  it  would  be  desirable  to  make  tests  for 
methyl-pentosans. 

Determinations  of  the  reducing  power  were  made,  as  shown  in  the 
following  table: 


SPECIES    OF    SEAWEED. 

SUGAR    AS    DEXTROSE. 

1  Hour. 

2  Hours. 

3  Hours. 

4  Hours. 

Limu  Manauea 

Per  cent. 

43.6 
36.0 

Per  cent. 

41.9 
58.4 
36.0 

Per  cent. 

44.6 
55.6 
34.0 

Per  cent. 

39.8 

Limu  Huna                

30.8 

Limu  Akiaki 

Slippery  Elm.  For  the  preparation  of  the  carbohydrate  which 
forms  the  mucUaginous  extract  of  shppery  elm  bark,  pieces  of  the 
latter  were  torn  into  narrow  strips  and  allowed  to  stand  over  night 
in  cold  water ,2  and  then  the  mucilage  expressed  by  squeezing  through 
gauze.  This  process  was  repeated  rnitil  the  bark  became  a  mass  of 
separate  fibers.  The  mucilaginous  principle  swells  in  cold  water  to 
a  transparent  jelly,  but  is  soluble  only  to  a  very  limited  extent.  It 
was  found  impossible  to  filter  it,  even  through  gauze,  and  therefore, 
although  it  contained  small  particles   from  the   disintegrated  bark 


1  For  method  cf .   Bull.  No.   107,  p.  55,  Bureau  of  Chemistry,  United  States 
Dept.  of  Agriculture. 

2  It  was  found  impossible  to  extract  the  mucilaginous  principle  in  hot  water. 


318  Mary  Davies  Swartz, 

fibers,  the  carbohydrate  was  precipitated  by  pouring  the  thick  slimy 
mass  into  about  six  times  its  volume  of  95  per  cent  alcohol.  After 
standing  some  hours,  a  transparent,  gelatinous  precipitate  settled  to 
the  bottom,  and  was  filtered  of!  through  several  thicknesses  of  gauze. 
Dehydrated  by  means  of  absolute  alcohol  and  ether,  it  formed  a  gray- 
ish-brown powder.  This  was  found  to  be  soluble  in  dilute  alkali,  and 
was  subsequently  purified  by  dissolving  in  1  per  cent  potassium  hy- 
droxide, filtering  through  cotton  and  reprecipitating  with  95  per  cent 
alcohol.  The  product  was  somewhat  Ughter  in  color  than  at  first, 
but  still  far  from  white.  It  was  soluble  in  hot  Fehling's  solution,  but 
precipitable  with  lead  acetate.  It  gave  no  color  with  iodine,  although 
a  small  amount  of  starch  was  present  in  the  original  bark. 

Furfurol  tests  were  faint  showing  only  traces  of  pentosans,  but  the 
yield  of  mucic  acid  was  large,  0.15  grams  of  mucic  acid  being  obtained 
from  ]  gram  of  the  air  dry  powder. 

The  products  of  hydrolysis  were  dextro-rotatory  and  contained 
no  fermenting  sugars.  Hence  this  preparation  consisted  chiefly  of 
galactan. 

A    MANNAN    PREPAEATION. 

Since  none  of  the  algae  which  form  the  basis  of  these  studies  yielded 
mannan,  save  Limu  Lipoa,  and  that  in  amounts  inadequate  for  the 
experiments  proposed,  this  hemicellulose  was  obtained  in  soluble 
form  from  salep.  Both  the  small,  horny  dried  tubers  and  the  gra3dsh- 
white  powder  made  from  them,  were  purchased  from  Schieffelein&  Co., 
New  York. 

A  preparation  of  pure  mannan  was  made  in  the  following  way: 
The  tubers  were  soaked  in  cold  water  24  hours,  washed  thoroughly 
and  ground  in  a  meat  chopper.  To  this  mass,  cold  water  was  added 
in  large  volume,  and  the  whole  allowed  to  stand  over  night,  then  the 
dissolved  mannan  filtered  ofi  through  gauze.  According  to  Hilger 
(254),  the  extract  made  in  this  way  should  contain  no  starch.  But 
when  the  tubers  are  heated  before  drying,  the  starch  is  made  soluble, 
and  in  this  instance  the  cold  water  extract  gave  a  blue  color  with 
iodine. 1  Hence  subsequent  extractions  were  made  with  hot  water  on 
a  water  bath,  for  several  hours.  The  salep  swells  very  much  in  water 
so  that  a  very  large  portion  was  required  to  get  the  mannan  all  into 


^Salep  tubers  purchased  since  this  work  was  done  yielded  only  a  trace  of  starch 
jn  the  cold  water  extract. 


Nutrition  Investigations.  319 

solution.!  Xhe  extracts,  strained  through  cheese  cloth,  were  digested 
24  hours  with  malt  diastase  to  free  from  starch,  then  concentrated  to 
a  thick  syrup  on  a  water  bath,  and  poured  into  three  times  their 
volume  of  95  per  cent  alcohol.  A  voluminous,  flocculent,  and  some- 
what fibrous,  snow-white  precipitate  formed,  which  was  filtered  off, 
pressed  free  from  alcohol,  redissolved  in  hot  water,  and  reprecipitated, 
(This  was  done  largely  to  free  it  from  sugar  produced  by  the  diges- 
tion of  the  starch.)  It  was  then  transferred  to  absolute  alcohol  and 
allowed  to  stand  three  or  four  days,  after  which  it  was  washed  with 
ether,  and  dried  in  a  vacuum  desiccator.  A  somewhat  coarse  white 
powder  resulted,  containing  6.94  per  cent  moisture  and  0.74  per  cent 
ash.2  It  swelled  up  very  readily  in  water,  but  dissolved  exceedingly 
slowly  to  a  colorless,  semi-transparent  mucilaginous  solution,  which 
did  not  reduce  Fehling's  solution,  and  examined  in  the  polariscope, 
after  clarification  with  alumina  cream,  appeared  optically  inactive. 
However,  on  reprecipitating  the  carbohydrate  with  alcohol,  and 
examining  the  alcohoUc  filtrate,  sugar  was  found  to  be  present  in 
small  amount.  A  solution  absolutely  sugar-free  became  optically 
active.  A  sample  in  which  the  sugar  had  been  removed  by  fermen- 
tation with  yeast,  was  used  to  determine  the  specific  rotation.  The 
following  results  were  obtained:  (1)  A  2  per  cent  solution  in  a  200 
mm.  tube  read  —1.59°;  applying  corrections  for  moisture  and  ash, 
[a]jy  =  —43.1°.  (2)  A  sample  containing  in  100  cc.  0.5868  grams 
mannan  dried  to  constant  weight  at  105°  C.  read  —0.48°;  corrected 
for  0.4  per  cent  ash,  [al^  =  —43.8°.  According  to  Thamm  (276), 
salep  extract  is  inactive.  In  the  above  experiments,  the  levo-rotatory 
nature  of  the  mannan  was  at  first  obscured  by  the  presence  of  traces 
of  reducing  sugar  formed  by  the  hydrolysis  of  the  starch,  which  could 
not  be  detected  by  testing  directly  by  Fehling's  solution.  Thamm, 
however,  in  several  ways  carefully  tested  salep  hydrolysis  products 
for  dextrose  with  negative  results,  so  that  the  only  way  to  account  for 
these  confhcting  results  seems  to  be  to  attribute  it  to  difference  in  the 
specimens  of  Orchis  which  furnished  the  mannan. 

Salep-extract  is  readily  precipitated  by  Fehhng's  solution  in  floccu- 
lent white  masses.  It  is  not  precipitated  by  lead  acetate  in  neutral 
solution  (nor,  according  to  Thamm  [276],  in  solutions  of  other  neutral 
salts),  but  is  precipitated  by  basic  lead  acetate. 

A  furfurol  test  was  faintly  positive,  verifying  the  report  of  traces  of 
pentosans  by  Tollens  and  Widtsoe  (163),  and  also  by  Thamm  (276). 

115  liters  of  water  to  100  grams  salep  powder,  according  to  Thamm  (276). 
^Thamm  found  0.483  per  cent. 


320  Mary  Dairies  Swartz, 

The  products  of  hydrolysis  were  dextro-rotatory  and  contained 
sugar  fermentable  with  yeast.  A  rich  yield  of  mannose-hydrazone 
was  obtained  with  phenyl-hydrazine  acetate,  melting  on  recrystalliza- 
tion  at  188°  C.  According  to  Thamm  (276),  salep  extract  yields  ex- 
clusively mannose  on  complete  hydrolysis. 

Hydrolyzed  for  three  hours,  the  reducing  power  of  this  mannan  was 
91.6  per  cent. 

Determinations  of  ash,  moisture,  starch,  and  mannan  were  made  on 
the  salep  obtained  in  the  form  of  a  powder.  Starch  and  mannan  were 
determined  as  follows:  1  gram  of  air  dry  powder  was  boiled  in  250  cc. 
water,  and  after  cooling  to  37.5°  C,  the  starch  hydrolyzed  with  malt 
diastase,  dialyzed  sugar-free.  The  solution  was  then  filtered,  con- 
centrated to  small  volume,  and  the  mannan  precipitated  with  absolute 
alcohol.  The  precipitate  was  filtered  off,  dissolved  in  a  little  water 
and  reprecipitated,  to  obtain  any  sugar  retained  in  the  first  precipi- 
tation. The  mannan  was  then  dried  at  100°  C.  and  weighed.  The 
filtrates  were  combined,  freed  from  alcohol,  hydrolyzed  with  2  per 
cent  hydrochloric  acid  45  minutes  to  convert  all  the  maltose  to  dextrose, 
and  sugar  determined  by  Allihn's  method.  The  results  of  these  analy- 
ses are  shown  in  the  following  table: 

Per  cent  Per  cent 

Moisture. 0.77         Starch 26.4 

Ash 8.9  Mannan 19.5 

According  to  Dragendorfi  the  composition  of  Orchis  tubers  is  as 
follows: 

Per  cent  Per  cent 

Starch 27.3  Protein 4.9 

Mucilage 48. 1  Cellulose 2.4 

Sugar 1.2 

Thamm  also  reports  a  yield  of  40^5  per  cent  mucilage  from  the 
salep  powder  used  in  his  investigations.  Hence  the  powder  used  in 
this  the  present  experiment  was  for  some  reason  very  deficient  in 
mannan. 

Its  reducing  power  w^as  as  follows: 

TIME   or  BOILING.  SUGAR    AS    DEXTROSE. 

Hours.  Per  cent 

2  74.2 

3  75.8 
5  75.8 

^Cited  in  the  National  Dispensatory  (1884),  also  by  Thamm  (276). 


Nutrition  Investigations.  321 

A  LEVULAN  PREPARATION. 

Commercial  Squills,  consisting  of  the  dried  and  broken  leaves  of 
the  bulbs  of  Scilla  maritima  (or  Urginea  Scilla  Stenh.)  jdeld,  as  dis- 
covered by  Schmiedeberg  (318),  the  levulan  sinistrin.  They  were 
finely  ground  in  a  coffee  mill,  and  the  sinistrin  prepared  according  to 
Schmiedeberg's  directions.  To  the  dry  powder  sufficient  water  was 
added  to  make  a  thin  cream,  and  then  a  saturated  lead  acetate  solu- 
tion until  further  addition  produced  no  precipitate.  To  the  clear, 
straw-colored  filtrate,  freed  from  lead  with  hydrogen  sulphide,  was 
added  freshly  prepared  milk  of  lime,  with  constant  stirring,  until  a 
somewhat  creamy  consistency  was  produced.  To  f aciUtate  the  for- 
mation of  sinistrin-calcium  carbonate,  this  mixture  was  concentrated 
on  the  water  bath  for  some  time  (as  suggested  by  Reidemeister)  [314]. 
The  precipitate  was  then  sucked  dry  on  a  Blichner  fimnel,  washed 
thoroughly  with  cold  water  (being  rubbed  up  in  a  mortar  for  the  pur- 
pose), again  sucked  dry,  rubbed  to  a  cream  with  water,  and  treated 
with  carbon  dioxide  imtil  the  fluid  was  no  longer  alkaline  to  litmus. 
After  heating  to  facilitate  the  complete  separation  of  the  calcium 
carbonate,  the  sinistrin  in  solution  was  filtered  off,  a  little  oxahc  acid 
carefully  added  to  remove  the  last  traces  of  lime,  and  the  solution  then 
decolorized  with  charcoal,  and  evaporated  to  a  syrup  at  a  temperature 
of  about  40°  C.  From  this  solution  the  sinistrin  was  precipitated  with 
95  per  cent  alcohol,  as  a  white  gummy  mass.  Transferred  to  abso- 
lute alcohol,  and  allowed  to  stand  24-36  hours  it  became  very  tenacious, 
but  on  longer  standing,  with  occasional  stirring,  it  grew  brittle,  and 
finally  crumbled  to  a  coarse  white  powder,  which  was  dried  in  a 
vacuum  desiccator.  This  material  was  readily  soluble  in  cold  water. 
(According  to  Schmiedeberg  [318],  even  solutions  of  20-30  per  cent 
are  not  syrup-like.)  It  gave  no  color  with  iodine,  did  not  reduce 
Fehling's  solution,  and  was  not  precipitated  by  it.  This  preparation, 
at  first,  contained  13  per  cent  moisture  and  0.76  per  cent  ash.  De- 
termination of  the  specific  rotation  then  gave  the  following  results: 
A  2  per  cent  solution  in  a  200  mm.  tube,  read  —1.32°;  corrected  for 
moisture  and  ash,  [a]D  =  -38.2°.  After  longer  standing  (three 
months)  over  sulphuric  acid,  the  moisture  content  was  4.8  per  cent, 
and  determination  of  specific  rotation  gave  the  following  results: 
A  1  per  cent  solution  in  a  200  mm.  tube,  read  -0.55°;  corrected  for 
moisture  and  ash,  [a]D  =  -29.1°.  Schmiedeberg  (318)  found  the 
average  for  [a]D  =  —41.4°,  and  Reidemeister  (314),  [a]D  =  —34.6°. 
It  is  impossible  to  account  for  these  differences.    Reidemeister  claims 


322 


Mary  Davies  Swartz, 


that  the  rotation  increases  on  standing,  but  in  these  solutions  there 
was  no  change  in  48  hours,  at  room  temperature. 

On  hydrolysis,  sinistrin  yields  a  levo-rotatory,  reducing  sugar,  fer- 
menting with  yeast.  Schmiedeberg  (318)  reports  this  as  a  mixture  of 
levulose  and  an  inactive  sugar,  but  Reidemeister  (314)  declares  that 
it  is  neither  a  mixture  of  levulose  and  an  inactive  sugar,  nor  of  levu- 
lose and  dextrose,  in  spite  of  the  fact  that  he  found  for  it  la]D=  —88°, 
while  for  levulose,  [a]D=  —106°,  a  difference  for  which  he  is  unable 
to  account. 

SUMMARY. 

The  composition  of  the  preparations  which  have  been  described  is 
best  shown  in  the  following  table: 


SOURCE  OF  MATERIAL. 


Dulse  (Rhodymenia  Palmata) 
Limu    Lipoa    {Haliseris  Par- 

dalis) 

Limu    Eleele     {Enteromorpha 

intestinalis) 

Limu    Pahapaha     {Ulva    lac- 

tuca,  etc.) 

Irish  Moss  {Chondrus  crispus) 
Limu  Manauea  {Gracilaria 

coronopifolia) 

Limu  Huna  {Hypnea  nidifica] 
Limu   Akiaki  {Ahnfeldtia  con- 

cinna) 

Limu     Uaualoli     {Gymnogon- 

grus) 

Limu    Kohu  {Asparagopsis 

sanfordiana) 

Slippery   Elm  iUlimis) 

Salep  {Orchis.) 

Squills  {Urginea  scilla)  [Sinis- 
trin]   


NATURE   OF  CARBOHYDRATES  PRESENT. 


Pentosans.     Galactan.      Mannan.       Levulan.       Dextran 


+ 

+ 

+ 

+ 
Trace 

+ 
+ 

+ 

+ 

+ 

Trace 


+ 

+ 
+ 

+ 

+ 

+ 
+ 


+ 


+ 


+ 


(Starch) 

+ 


The  foregoing  observations  correspond  with  those  of  Konig  and 
Bettels  (8),  in  that  the  marine  algae  all  yield  pentosans,  and  fre- 
quently galactans.  The  gelatinizing  principle  in  every  case  appears 
to  be  due  to  the  galactan  groups.     No  specific  tests  have  been  applied 


Nutrition  Investigations. 


323 


for  fructose,  the  polysaccharide  of  which  also  appears  to  be  common 
in  algae,  but  the  absence  of  fermenting  sugar  in  all  the  algae  except 
Limu  Lipoa,  indicates  that  if  present,  it  is  in  too  small  amount  to  be 
detected  in  the  hydrolysis  products  of  5-10  grams  of  crude  material. 
The  reducing  power  has  been  determined  on  each  substance  used  in 
feeding  experiments;  the  results  of  all  determinations  are  summarized 
in  the  following  table: 


SUBSTANCE. 


SUGAR   AS   DEXTROSE   ASTER    BOILING. 


1  Hour.'2  Hovirs.  3  Hours. 


4  Hours.  5  Hours.  6  Hours.  8  Hours 


Dulse 

Limu  Lipoa 

Limu  Eleele 

Limu  Pahapaha 

Irish  Moss 

Limu  Manauea 

Limu  Huna 

Limu  Akiaki 

Salep  (Powder) 

Salep  (Pure  mannan) 


[Per  cent.  Per  cent. 

I  87.2 

I 

I  16.8 
:  28.8 

.  45.6 

I  41.9 

43.6  !  58.4 

36.0     36.0 

1 
74.2 


Per  cent.  Per  cent  J  Per  cent. 

87.3  89.4  !  89.5. 

14.3  I  14.7  I 

16.9  !  18.1  j  16.8 

i  31.8  i 


48.6  1  45.8 
44.6  '  39.8 


55.6  i  30.6 


34.0 

75.8 
91.6 


Per  cent. 
12.9 


Per  cent. 

12.8 


75.8 


Bacteriological  Investigations. 


INTRODUCTION. 


It  is  an  accepted  fact  that  even  cellulose,  with  its  high  powers  of 
resistance,  is  to  some  extent  decomposed  in  the  alimentary  tract  by 
bacteria.  It  is  therefore  reasonable  to  expect  that  the  less  resistant 
hemicelluloses  will  also  be  attacked  and  decomposed  by  bacteria. 
The  object  of  these  experiments  has  been  to  throw  some  Hght  on  the 
problem  as  to  what  organisms  are  most  likely  to  effect  such  a  decom- 
position, and  whether  there  is  an  appreciable  production  of  sugar  as 
a  result  of  bacterial  activity.  The  four  classes  of  hemicelluloses  under 
special  investigation  have  been  represented  by  the  following  sub- 
stances : 


Pentosans Dulse. 

/  Irish  Moss. 
'  \  Limu  Manauea. 


Galactans . 


Mannans Salep. 

Levulans Sinistrin. 


324  Mary  Davies  Swartz, 

Both  aerobic  and  anaerobic  cultures  have  been  made,  in  neutral, 
faintly  alkaline,  and  faintly  acid  reaction,  with  solutions  made  from 
the  carbohydrates  alone,  and  with  the  addition  of  small  amounts  of 
such  nutrients  as  beef  extract  or  peptone  to  facilitate  the  growth  of 
the  organisms. 

Anaerobic  cultures  in  test  tubes  have  been  made  by  the  Wright 
method ;  anaerobic  ciiltures  in  Erlenmeyer  flasks,  by  passing  a  stream 
of  hydrogen  through  for  half  an  hour,  and  then  sealing  hermetically. 

The  aerobes  which  have  been  employed  all  occur  in  the  human 
digestive  tract.  Both  aerobic  and  anaerobic  cultures  from  the 
faeces  of  human  subjects  have  also  been  used,  in  conjunction  with 
soil  bacteria  from  street  sweepings. 

Tests  for  the  presence  of  reducing  sugar  have  been  made  by  pre- 
cipitating the  carbohydrates  in  solution  with  absolute  alcohol,  evapor- 
ating the  alcoholic  extract  to  dryness,  taking  up  the  residue  in  2  or 
3  cc.  of  water,  and  boiling  two  minutes  with  Fehling's  solution. 

Suitable  controls  have  been  used  in  all  cases. 

TRIALS  WITH  PURE  CULTURES  OE  AEROBES. 

One  per  cent  solutions  of  the  preparations  from  dulse,  Irish  moss  and 
salep,  neutral,  acid,  and  alkaline  in  reaction,  and  consisting  of,  (1) 
pure  carbohydrate;  (2)  carbohydrate  plus  J  per  cent  beef  extract  and 
f  per  cent  sodium  chloride;  (3)  carbohydrate  plus  1  per  cent  peptone 
and  i  per  cent  sodium  chloride,  have  been  used  as  culture  media. 
Five  cc.  portions  of  each  of  these  solutions  were  placed  in  test-tubes 
with  a  pipette,  and  inoculated  with  the  following  organisms:  B.  Coli 
communis,  B.  Pyocyaneus,  B.  Prodigiosus,  B.  Proteus  vulgaris, 
B.  Pyogenes  foetidus. 

To  approximate  the  conditions  in  ordinary  digestion  of  these  car- 
bohydrates, they  were  incubated  for  three  days  at  a  temperature  of 
37.5°  C.  At  the  end  of  this  time,  nearly  all  gave  evidence  of  some 
bacterial  growth.  Salep-peptone  cultures  of  B.  Pyocyaneus  showed 
a  brilliant  green;  salep  solutions  containing  B.  Pyogenes  foetidus, 
and  B.  Coli  in  alkaline-beef  extract  media,  had  changed  from  trans- 
parent colorless  solutions  to  an  opaque  white  jelly  insoluble  in  water. 

The  carbohydrates  were  then  precipitated  with  alcohol,  and  after 
standing  several  days  were  compared  with  controls  similarly  prepared, 
to  see  whether  any  change  could  be  observed  in  the  nature  or  amount 
of  carbohydrate.  The  results  were  in  all  cases  negative.  These  pre- 
cipitates were  then  transferred  to  small  folded  filter  papers  of  uniform 


Nutrition  Investigations.  325 

weight,  previously  prepared.  The  alcohoHc  filtrates  were  tested  for 
sugar;  the  precipitates  were  dried,  and  their  weight  compared  with 
that  of  the  control.  It  was  thought  that  this  rather  crude  method 
would  show  whether  any  considerable  amount  of  the  carbohydrate 
had  disappeared.  The  results  were  so  largely  negative  that  weighings 
of  every  precipitate  were  not  made.  There  seemed  to  be  a  slight  loss 
of  dulse,  in  some  of  the  cultures  of  B.  Proteus  vulgaris,  B.  Pyogenes 
foetidus,  and  B.  Coli  communis,  but  repetition  of  these  experiments 
allowing  the  organisms  in  question  to  grow  two  weeks,  not  only  in 
dulse  but  also  in  salep  media,  did  not  justify  any  conclusion  that  an 
appreciable  amount  of  carbohydrate  had  disappeared. 

All  tests  for  reducing  sugar  were  negative. 

Four  per  cent  solutions  of  Irish  moss,  and  two  per  cent  solutions 
of  limu  manauea  were  then  prepared,  with  reactions  and  additions  of 
nutrient  material  as  described  in  the  first  series  of  experiments.  These 
formed  firm  jelHes,  which  were  used  to  study  the  possibility  of  lique- 
faction or  gas  formation.  Stab  cultures  were  made,  and  grown  at  a 
temperature  of  25°-30°  C.  for  one  to  three  weeks.  No  hquefaction 
or  gas  formation  was  observed  in  any  case. 

TRIALS  WITH  MIXTURES  OF  AEROBES. 

Mixtures  of  B.  Pyocyaneus,  B.  Prodigiosus,  B.  Proteus  vulgaris, 
and  B.  Pyogenes  foetidus,  were  used,  also  mixtures  of  faecal  and  soil 
bacteria.  These  were  first  inoculated  into  nutrient  bouillon,  the 
former  from  pure  cultures,  the  latter  from  human  faeces  and  street 
sweepings,  and  incubated  24  hours.  Five  cc.  portions  of  these  cultures 
were  then  introduced  into  50  cc.  of  neutral  solutions  of  each  of  the 
different  carbohydrates,  in  small  Erlenmeyer  flasks,  and  these  cul- 
tures allowed  to  grow  for  four  weeks  at  37.5°  C.  At  the  end  of  this 
time,  no  marked  change  had  taken  place  save  in  the  salep  culture  of 
B,  Pyocyaneus,  B.  Proteus  vulgaris,  B.  Pyogenes  foetidus  and  B. 
Prodigiosus.  This  had  changed  from  a  colorless,  semi-transparent, 
shghtly  mucilaginous  fluid,  to  a  firm,  white  opaque  jelly,  insoluble 
in  water,  but  readily  soluble  in  dilute  alkali;  a  phenomenon  already 
observed  with  this  carbohydrate  in  cultures  of  B.  Coli  communis  and 
B.  Pyogenes  foetidus.  No  liquefaction  had  taken  place  with  Irish 
moss  nor  limu  manauea. 

The  carbohydrates  were  then  precipitated  with  alcohol,  the  alco- 
holic extracts  tested  for  sugar,  and  the  precipitates  hydrolyzed  by 
boiling  with  2  per  cent  hydrochloric  acid,  neutrahzed,  made  up  to  a 


326 


Mary  Davies  Swartz, 


definite  volume,  and  examined  in  a  polariscope.  The  results  of  these 
experiments  are  shown  in  the  following  table.  Mixtures  of  B.  Pyo- 
cyaneus,  B.  Prodigiosus,  B.  Proteus  vulgaris  and  B.  Pyogenes  foetidus 
are  designated  A,  and  mixtures  of  faecal  and  soil  bacteria,  B. 


BACTERIAL 

REDUCTION 
OF 

ROTATION   AFTER  HYDROLYSIS. 

CULTURE         '        FEHLING'S 
SOLUTION. 

Experiment. 

Control. 

Dulse 

B 
A 
B 

:::::+: 

+  0.13° 
+  0.20° 

+0.27° 

Not  determined. 

Not  determined. 

+0.17° 

-0.97° 

+0.20° 

Irish  Moss 

Irish  Moss 

+  0.20° 

Limu  Manauea  .... 

Salep 

Salep 

Sinistrin 

A 
A 
B 
A 

+0.20° 
—0.97° 

The  action  of  putrefactive  organisms  upon  the  dulse  preparation 
was  also  studied,  according  to  the  method  used  by  Slowtzofi  (154) 
in  the  case  of  xylan.  One  hundred  grams  of  chopped  lean  beef  and 
10  grams  of  sodium  carbonate  were  added  to  1  liter  of  water,  and 
the  mixture  allowed  to  stand  in  a  warm  place  for  three  days.  Two 
himdred  and  fifty  cc.  were  then  removed  for  a  control,  and  to  the 
remainder  0.5  gram  of  dulse  was  added.  This  solution  gave  a  strong 
pentosan  reaction;  the  control  was  pentosan-free.  The  two  solutions 
were  put  in  a  warm  place,  and  tested  daily  for  pentosans.  After  five 
days'  digestion,  the  reaction  of  the  dulse  solution  was  very  much 
fainter  than  at  first,  but  it  did  not  entirely  disappear  till  the  twelfth 
or  thirteenth  day.  Slowtzofi  found  that  xylan  disappeared  in  nine 
or  ten  days,  but  his  solution  was  kept  at  a  temperature  of  40°  C, 
while  these  mixtures  remained  at  a  temperature  of  from  30°  to  35°  C, 
a  condition  less  favorable  for  rapid  decomposition. 

Solutions  of  Irish  moss  were  digested  with  faecal  mixtures  in  the 
following  manner:  Human  faeces  were  rubbed  to  a  mud  with  water. 
Ten  cc.  portions  of  this  material  were  added  to  flasks  containing  50  cc. 
of  a  1  per  cent  "moss"  solution,  and  allowed  to  digest  in  a  warm  place 
for  24  hours.  A  portion  of  water  inoculated  in  the  same  way  was 
used  as  a  control.  Small  portions  of  these  solutions  were  then  evap- 
orated nearly  to  dr3aiess,  extracted  with  alcohol,  and  tested  for  reduc- 
ing sugar.     The  results  were  wholly  negative. 

That  limu  manauea  is  not  entirely  resistant  to  the  action  of  putre- 
fying organisms  is  shown  by  the  following:  A  solution  was  made 


Nutrition  Investigations.  327 

up  to  contain  2  per  cent  of  the  air  dry  extract,  1  per  cent  peptone, 
J  per  cent  beef  extract  and  \  per  cent  sodium  chloride.  This  could  be 
filtered  through  paper  only  on  a  hot,  water- jacketed  funnel,  from 
which  it  dropped  as  a  clear,  amber-colored  jelly.  After  standing 
imsterilized  over  night  in  a  warm  room,  this  was  found  to  be  entirely 
broken  up  by  the  formation  of  gas  throughout  the  whole  mass.  The 
reaction,  which  had  been  neutral,  was  now  acid  to  litmus.  This 
material  was  placed  in  a  flask  and  allowed  to  stand  for  two  months, 
at  the  end  of  which  time,  the  greater  portion  was  Uquefied,  the  former 
lumps  of  jelly  being  reduced  to  smaU  particles  distributed  throughout 
the  liquefied  portion.  Alcoholic  extracts  did  not  reduce  Fehling's 
solution.  A  sterile  preparation  of  the  plain  manauea  extract  in  test 
tubes  was  inoculated  with  some  of  this  material,  but  without  produc- 
ing the  same  striking  results.  There  were  evidences  of  growth,  but 
none  of  liquefaction  or  gas  formation,  in  the  course  of  two  weeks. 

TRIALS  WITH  ANAEROBES. 

The  action  upon  Irish  moss  of  pure  ciiltures  of  the  powerful  putre- 
factive organisms  B.  Putrificus,  Bienstock,  B.  Mahgni  oedematis, 
and  B.  Anthracis  symptomatici,  was  tried  in  the  following  way.  A 
4  per  cent  solution  of  the  moss  was  prepared,  which  would  not  become 
Hquefied  at  a  temperature  of  30°-35°  C.  From  this  material  culture 
media  were  prepared,  neutral,  alkaline,  and  acid  in  reaction,  using 
the  solution  plain,  and  with  the  addition  of  |  per  cent  beef  extract 
and  \  per  cent  salt,  or  1  per  cent  peptone  and  \  per  cent  salt.  Test 
tubes  were  inoculated  from  fresh,  active  cultures,  and  the  organisms 
allowed  to  grow  for  one  to  three  weeks,  being  examined  at  first  daily, 
and  later  every  three  or  four  days,  for  liquefaction  and  gas  formation. 
The  results  were  negative  in  all  cases,  save  that  in  the  peptone  media 
an  occasional  small  bubble  was  seen,  mth  cultures  of  the  bacilli  of 
mahgnant  oedema  and  symptomatic  anthrax.  However,  the  same 
phenomena  were  observed  in  peptone-agar  tubes  used  as  controls. 

Mixtures  of  B.  Anthracis  symptomatici  and  B.  MaHgni  cedematis 
were  tried  upon  solutions  of  dulse,  Irish  moss,  salep  and  sinistrin,  in 
the  following  way:  Small  Erlenmeyer  flasks  containing  50  cc.  of 
1  per  cent  solutions  of  each  of  these  carbohydrates,  and  5  cc.  of  ordi- 
nary nutrient  bouillon,  were  inoculated  with  fresh  ciiltures  of  these 
organisms,  rendered  anaerobic,  and  incubated  for  four  weeks  at  37.5** 
C.  On  inspection,  no  change  was  apparent.  The  carbohydrates 
were  removed,  the  alcoholic  extracts  examined  for  reducing  sugar,  and 


328 


Mary  Davies  Swartz, 


the  carbohydrate  residues  hydrolyzed  and  examined  in  the  polari- 
scope,  as  in  similar  trials  with  aerobes.  The  results  are  shown  in 
the  following  table: 


NAME    OF    SUBSTANCE. 


reduction  of 
eehling's 
solution. 


ROTATION    AFTER    HYDROLYSIS. 


Experiment. 


Control. 


Dulse 

Irish  Moss 

Salep 

Sinistrin . . 


Lost  by  laccident 
+  0.24°     I     +0.20° 
+  0.13°         +0.20° 
-  0.27°         -  0.97° 


Mixtures  of  soil  and  faecal  bacteria  were  also  tried,  the  experiments 
being  carried  out  just  as  described  for  mixtures  of  the  bacilli  of  symp- 
tomatic anthrax  and  malignant  oedema.  The  results  are  shown  in 
the  following  table: 


reduction  of 
feeling's 
solution. 

ROTATION  AFTER  HYDROLYSIS. 

NAME    OF    SUBSTANCE. 

Experiment. 

Control. 

Dulse 

Irish  Moss     

+ 

+  0.13° 
+  0.20° 

+  0.03° 

+  0.20° 

+  0.20° 

Salep 

+  0.20° 

DISCUSSION    AND    SUMMARY. 

It  seems  reasonable  to  expect,  that  if  the  hemicelluloses  used  in 
these  trials  were  readily  attacked  by  micro-organisms,  there  would  have 
been  some  evidence  of  change  in  three  days,  if  conditions  for  growth 
were  favorable  as  regards  reaction  and  temperature;  but  although  the 
concentration  of  the  solutions  was  moderate,  the  reaction  varied, 
and  temperature  37.5°  C,  results  were  negative,  even  in  the  cases 
where  nutrients  were  added  to  facilitate  bacterial  growth.  Apparently 
all  of  the  material  was  recovered  in  unaltered  condition,  save  in 
certain  instances  where  salep  underwent  an  insoluble  modification. 

In  trials  where  the  cultures  were  allowed  to  grow  from  one  to  three 
weeks,  no  dilf  erence  in  the  results  could  be  detected,  by  the  methods 
employed.  In  solid  media  there  was  no  liquefaction  and  practically 
no  gas  formation,  except  in  the  case  of  the  peptone-beef  extract 
preparation  of  limu  manauea,  on  exposure  to  the  air. 


Nutrition  Investigations. 


329 


Marked  evidences  of  change  were  observed  in  one  trial  with  a  putre- 
factive mixture  (on  diilse),  and  in  some  of  the  four- week  cultures. 

Irish  moss  was  the  most  thoroughly  investigated  and  proved  the 
most  resistant.  In  the  long  experiments  (4  weeks)  where  the  other 
carbohydrates  suffered  more  or  less  change  this  one  remained  appar- 
ently unaltered.  The  results  of  this  series  are  summarized  in  the 
following  table: 

Irish  Moss. 


CULTURES   USED. 


reduction  of 
fehxing's 
solution. 


ROTATION   OF   UNALTERED   CAR- 
BOHYDRATE AFTER  HYDROLYSIS. 


Irish   Moss. 


Control. 


Mixture  of  Pure  Aerobes . 


Mixture    of  Faecal   and  Soil  Bacteria 
(aerobic) 

Mixture      of      Bacilli     of     Malignant 
Oedema  and  S3Tnptomatic  Anthrax .  . 

Mixture  of    Faecal  and  Soil    Bacteria 
(anaerobic) 


+  0.20°     I      +0.20° 

i 
+  0.27°     '      +0.20° 


+  0.24'= 


+  0.20° 


+  0.20°    I       +0.20° 


The  single  experiment  with  the  galactan,  limu  manauea,  imder  the 
same  conditions,  with  the  mixture  of  pure  aerobes,  gave  similar 
results,  but  the  fact  that  Uquefaction  occurred  in  the  peptone-beef 
extract  culture  medium  after  exposure  to  the  air,  shows  that  general 
conclusions  as  to  the  behavior  of  galactans  cannot  be  drawn  from 
study  of  a  single  representation  of  the  class.  We  have,  however, 
further  proof  that  the  galactans  are  not  easily  decomposed  by  bacteria^, 
in  the  fact  that  aqueous  solutions  of  all  the  galactans  included  in  the 
present  series,  could  be  left  several  days  in  the  warm  atmosphere  of 
the  laboratory  without  any  apparent  change  taking  place;  and  in  the 
fact  that  agar-agar,  so  widely  used  in  bacteriological  laboratories 
on  account  of  its  indifference  to  bacterial  action,  is  a  member  of  the 
galactan  group.  It  has  been  suggested^  that  extracts  of  other  sea- 
weeds might  prove  good  substitutes  for  agar-agar  as  culture  media, 
if  fully  investigated.  So  far,  the  greatest  objection  to  use  of  Irish 
moss  in  this  way  is  that  it  tends  to  Kquefy  at  body  temperature; 
strong  solutions  (4  per  cent)  can,  however,  be  kept  fairly  firm  at  a 


iCf.  Reed  (18). 


330 


Mary  Davies  Swartz, 


temperature  of  30°  C.  The  extract  of  limu  manauea  is  free  from 
these  objections,  but  extensive  experiment  is  still  necessary  to  demon- 
strate its  powers  of  resistance. 

The  soluble  dulse  pentosan  is  certainly  decomposed  not  only  by 
putrefactive  organisms  under  the  most  favorable  conditions  {e.g.,  in 
meat  mixtures),  but  by  aerobes  and  anaerobes  in  solutions  where  the 
carbohydrate  is  the  chief  source  of  nutriment.  The  results  of  the 
four  weeks'  digestions  are  summarized  in  the  following  table: 


Dulse. 


CULTURES    USED 

REDUCTION 
OP 

fehling's 
solution. 

ROTATION  OP  UNALTERED 

CARBONTDRATE   APTER 

HYDROLYSIS. 

Dulse. 

Control. 

Mixture  of  Faecal   and   Soil  Bacteria 
(aerobic)                  

+  0.13° 

(Lost  by 
Accident) 

+0.13° 

+0.20° 

Mixture   of  the  Bacilli  of  Malignant 
Oedema  and  Symptomatic  Anthrax.  . 

Mixture   of   faecal    and   Soil  Bacteria 
(anaerobic) 

+0.20° 
+0.20° 

In  the  present  studies,  this  pentosan  stands  second  to  the  galactans 
in  degree  of  resistance. 

Sawamura  (267)  thought  that  he  observed  a  slight  hydrolysis  of 
mannan  by  B.  Prodigiosus,  an  observation  which  has  not  been  verified 
in  these  experiments.  No  reducing  substance  was  detected  in  the 
three-day  cultures  nor  the  four-weeks  cultures,  in  which  this  organism 
was  present.  The  opaque  jelly,  insoluble  in  water,  formed  from  salep 
by  the  action  of  B.  Coli  communis,  B.  Prodigiosus,  and  mixed  cultures 
containing  these  organisms,  resembles  an  intermediary  product  of 
the  acid  hydrolysis  of  salep-mannan  described  by  Thamm  (276). 
He  isolated  and  examined  two  such  products,  one  forming  an  opales- 
cent solution  in  water,  the  other  insoluble,  but  passing  over  into  the 
soluble  form  by  treatment  with  dilute  alkali;  both  were  anhydrides 
of  mannose.  It  seems  reasonable  to  inquire  whether  this  insoluble 
material  produced  by  bacterial  action  may  not  be  regarded  as  an 
early  stage  in  the  hydrolysis  of  the  carbohydrate  under  consideration, 
especially  in  view  of  the  fact  that  in  all  the  other  four-week  trials  a 
very  definite  reduction  of  Fehling's  solution  was  noted,  corresponding 


Nutrition  Investigations. 


331 


in  strength  with  the  loss  of  unaltered  carbohydrate,  as  shown  in  the 
following  summary: 

Salep. 


CTXLTUKES  USED. 


REDUCTION  OP 
rETTT.TNG  's 
SOLUTION. 


ROTATION  OF  UNALTERED    CARBO- 
HYDRATE APTER  HYDROLYSIS. 


Mixture  of  Pure  Aerobes !  (Insoluble 

I        jelly) 
Mixture   of   Faecal  and  Soil    Bacteria  ' 

(aerobic) + 


Salep. 


Not  det  ermined 
+  0.17°     i      +0.20° 


Control. 


Mixture   of    the  Bacilli  of   Malignant 
Oedema  and  Symptomatic  Anthrax .  . 

Mixture  of    Faecal  and  Soil  Bacteria 
(anaerobic) 


+ 


+ 


+  0.13° 
+  0.03^ 


+  0.20° 


+  0.20° 


These  experiments  give  some  grounds  for  expecting  the  hydrolysis 
of  salep  in  the  aUmentary  tract,  through  the  action  of  bacteria. 
Two  experiments  with  sinistrin  gave  the  followmg  results: 


Sinistrin. 


reduction  of 
fehling's 

SOLUTION. 

ROTATION  OF  UNALTERED  CAR- 
BOHYDRATE AFTER  HYDROLYSIS. 

CULTURES  USED. 

Sinistrin. 

Control. 

+ 

-0.97° 
-0.27° 

-0.97° 

Mixture  of  Bacilli  of  MaHgnant  Oedema 
and   Symptomatic  Anthrax 

-0.97° 

Sinistrin  is  therefore  hydrolyzed  by  the  anaerobic  putrefactive 
organisms,  but  further  experiments  are  necessary  to  determine  how 
readily  this  change  takes  place. 


Physiological  Investigations. 

In  the  physiological  experiments,  attempts  have  been  made  to 
answer  the  following  questions:  (1)  To  what  extent  are  hemiceUuloses 
digested  by  animal  and  vegetable  enzymes?  (2)  Can  they  be  ab- 
sorbed and  utUized  without  intervention  of  the  alimentary  tract? 


332  Mary  Davies  Swartz, 

(3)  Do  they  reappear  in  the  faeces  after  administration  per  os?  The 
various  experiments  will  accordingly  be  discussed  in  these  three 
groups:  (1)  Trials  with  Enzymes;  (2)  Parenteral  Trials;  (3)  Feeding 
Experiments. 

TRIALS   WITH   ENZYMES. 

Approximately  1  per  cent  solutions  of  the  various  hemicelluloses 
(with  the  exception  of  Limu  Lipoa,  which  was  finely  ground  and  sus- 
pended in  water),  have  been  digested  for  24  hours  at  37.5°  C.  in  the 
presence  of  toluene,  with  the  following  enzymes:  (1)  Filtered  human 
saliva.  (2)  Malt  diastase,  dialyzed  sugar-free.  (3)  "Taka"  dias- 
tase {Eurotium  oryzae).  (4)  Chloroform  extract  of  pig's  pancreas. 
(5)  Fresh  pancreatic  juice  of  dogs.  (6)  Chloroform  water  extract 
of  dog's  intestines.     (7)  Glycerol  extract  of  pig's  stomach. 

Digestions  have  also  been  made  with  0.2  per  cent  hydrochloric  acid, 
to  determine  whether  any  of  the  action  of  the  artificial  gastric  juice 
might  be  due  to  the  acid  present.  The  activity  of  the  amylolytic 
enzymes  has  always  been  tested  first  with  starch  paste,  and  that  of 
the  gastric  extract  with  fibrin.  Boiled  controls  have  been  employed 
in  every  instance,  and  all  trials  have  been  made  in  duplicate. 

Tests  for  reducing  sugar  have  been  conducted  in  the  following 
manner:  At  the  end  of  24  hours  the  solutions  were  evaporated  to 
thick  syrups  on  the  water  bath,  to  free  from  toluene  and  to  concen- 
trate so  that  the  undigested  hemicelluloses  could  be  readily  precipi- 
tated by  absolute  alcohol.  The  alcoholic  extracts  were  filtered  ofi 
and  evaporated  to  dryness;  the  residues  were  taken  up  in  a  few  drops 
of  water  and  tested  for  sugar  with  FehHng's  solution.  The  results 
of  all  digestion  trials  are  shown  in  the  table  on  opposite  page. 

PARENTERAL    INJECTIONS. 

Methods  and  Technique. 

Small  dogs  were  used  for  all  injections,  after  a  confinement  in  cages 
long  enough  to  obtain  samples  of  normal  urine.  The  carbohydrates 
employed  in  these  experiments  were  preparations  of  dulse,i  Irish  moss,^ 
salep,^  and  sinistrin.*    They  were  introduced  suhcutaneously ,  by  means 

iCf.  p.  303. 
2Cf.  p.  308. 
3Cf.  p.  312. 
«Cf.  p.  315. 


+  I  +  + 


+  ^o 


+ 


+    + 


+ 


+ 


+ 


02 

c3 

CO  + 

.2 


O     tn     C     pj     cd     "     rt 

;3  1  S  w  <  t^  fc^ 

0     -,    :i    0    ^    ^    7i 

s  :i  s  s  s  a  s 
p  vii  ;j  13  '^13  P  ^ 

w 


OOOUUOOOOC    -72 


334  Mary  Davies  Swariz, 

of  a  syringe,  or  intraperitoneally,  by  means  of  a  needle  and  burette 
with  pressure-bulb  attached,  always  under  aseptic  conditions.  After 
receiving  injections,  the  animals  were  replaced  in  cages,  and  the 
urine  collected  under  toluene.  The  excess  of  toluene  was  removed, 
at  the  time  of  examination,  by  means  of  a  separatory  funnel,  and  the 
urine  measured,  filtered,  and  tested  for  reducing  substances  with 
Fehling's  solution. 

Qualitative  tests  for  the  carbohydrates  were  made  in  the  following 
manner:  (l)  for  dulse  and  salep,  by  boiling  a  few  drops  of  urine  with 
Fehling's  solution,  from  which  these  hemicelluloses  were  precipitated 
in  fine  white  flocks,  even  if  only  traces  were  present;  (2)  for  Irish 
moss,  by  the  reduction  of  Fehling's  solution  after  hydrolysis  of  the 
urine  with  dilute  hydrochloric  acid;i  (3)  for  sinistrin,  by  the  marked 
increase  in  the  levo-rotation  of  the  urine. 

Isolation  of  the  carbohydrates  was  accomplished  by  freeing  the 
urine  from  inorganic  salts  with  lead  acetate,  removing  the  excess  of 
lead  with  hydrogen  sulphide,  and  concentrating  the  salt-free  solutions 
to  a  small  volume.  Dulse  and  Irish  moss  were  then  precipitated  with 
absolute  alcohol;  salep  with  alcohol  or  Fehling's  solution;  sinistrin 
with  milk  of  lime,  being  freed  from  its  calcium  compound  by  the 
method  used  in  its  preparation. ^ 

These  substances  were  identified  as  carbohydrates,  by  their  yield- 
ing reducing  sugar  on  hydrolysis;  salep  and  sinistrin  were  further 
identified  by  their  levo-rotation,  Irish  moss  by  testing  for  mucic  acid, 
and  dulse  by  testing  for  furfurol. 

Quantitative  determinations  of  dulse,  salep  and  sinistrin  were  made 
by  polariscopic  examination  in  a  200  mm.  tube,  all  samples  of  urine 
being  clarified  with  equal  volumes  of  alumina  cream.  A  satisfactory 
quantitative  method  for  the  determination  of  Irish  moss  was  not 
developed.  It  proved  impossible  to  estimate  any  of  these  carbohy- 
drates quantitatively  by  the  method  of  acid  hydrolysis.  In  some 
instances,  especially  with  Irish  moss,  a  trace  of  reduction  was  ob- 
tained, but  in  most  cases,  the  results  were  negative,  although  the  hemi- 
cellulose  was  known  to  be  present.^ 

^Trial  was  made  of  Bauer's  method  (Zeitschrift  fiir  physiologische  Chemie,  51, 
p.  158,  1907)  of  determining  galactose  in  urine  as  mucic  acid,  by  concentrating 
100  cc.  of  urine  with  25-35  cc.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  to  a  volume  of 
20  cc,  but  owing  probably  to  the  low  percentage  of  galactose  from  the  small  amount 
of  Irish  moss  present,  this  test  was  unsatisfactory. 

2Cf.  p.  315. 

^Samples  were  removed  and  tested  every  half  hoiir  for  2^  hours.  At  the  end  of 
1  hour  they  were  usually  neutral,  or  slightly  alkaline  in  reaction.     Addition  of  suf- 


Nutrition  Investigations. 


335 


INJECTIONS    OF  DUXSE. 

1.  Subcutaneous. 

A  dog  weighing  11  kg.  received  60  cc.  of  a  dulse  solution  contain- 
ing 0.9  grams  of  pure  substance.  No  reduction  of  Fehling's  solution 
was  observed  at  any  time.  The  time  and  rate  of  dulse  excretion  are 
shown  in  the  following  table : 

Examination  of  Urine. 


ESTIMATED  EXCRETION 
OF  DULSE.* 


February  1,  12:30  P.M 

February  1,    1  P.M Injection 

February  2,  10  A.M ''■  226 

February  3,  10  A.M 250 

February  4,  10  A.M I  150 

February  5,  10  A.M '  210 

February  6,  10  A.M :  310 

February  7,  10  A.M j 


-0.14°t 

-  0.62° 
-0.55° 
-0.41° 
-0.34° 

-  0.28° 

-  0.20° 


Total. 


Grams. 


0.61 
0.57 
0.21 
0.21 
0.04 


1.64 


2.  Intraperitoneal. 

The  same  dog  received  in  this  experiment  75.6  cc.  of  a  dulse  solu- 
tion containing  1.4  grams  of  pure  substance.  No  reduction  of  Feh- 
ling's solution  was  observed  before  or  after  the  injection.  The  time 
and  rate  of  dulse  excretion  are  shown  in  the  following  table: 

Examination  of  Urine. 


ROTATION. 


ESTIMATED  EXCRETION 
OF  DIILSE.* 


December  3,    2  P.M | 

December  3,    3  P.M i    Injection 

December4,  lOA.M |  133 

December5,  lOA.M I  200 

December  5,  12  M '  115 

December  6  and  7 |  383 

December  8,  10  A.M 520 

December  9,  10  A.M 350 


-0.14°t 


Grams. 


-  0.62° 

0.36 

-  0.52° 

0.42 

-  0.28° 

0.05 

-0.48° 

0.69 

-0.28 

0.24 

-0.20 

Total 

1.76 

ficient  hydrochloric  acid  to  make  the  strength  2  per  cent  caused  no  subsequent 
production  of  sugar.- 

*  All  readings  have  been  taken  on  the  Ventzke  scale,  and  calculated  as  angular  degrees. 

t  Estimating  normal  rotation  of  urine  as  — 0.17°  (average). 


336 


Mary  Davies  Swartz, 


In  both  these  experiments,  the  presence  of  dulse  was  readily  detected 
by  Fehling's  solution  in  every  urine  which  showed  a  high  rotation. 
From  the  samples  of  the  first  48  hours  after  injection,  a  considerable 
amount  was  isolated  and  identified  as  carbohydrate.  It  is  evident 
that  the  excretion  of  this  pentose-carbohydrate  is  gradual,  commenc- 
ing soon  after  the  injection,  and  continuing  from  four  to  five  days. 
WTiile  any  quantitative  estimate  of  the  amount  excreted,  based  on  the 
changes  in  rotation,  is  subject  to  a  high  percentage  of  error,  owing  to 
normal  fluctations  in  the  rotation  of  the  urine,  as  well  as  to  analyt- 
ical discrepancies  unavoidable  in  dealing  with  solutions  containing 
only  minute  quantities  of  the  substance  under  investigation,  it  is  evi- 
dent that  most  of  the  dulse  must  have  been  excreted,  and  that,  too, 
without  any  essential  change  in  character. 

INJECTIONS   or  IRISH  MOSS. 

1.  Subcutaneous. 


A  dog  weighing  9.4  kg.  received  100  cc.  of  Irish  moss  solution,  con- 
taining 1.5  grams  of  dry  substance.  No  reducing  substance  occurred 
in  the  urine.  Changes  in  rotation,  due  to  the  injection,  are  shown  in 
the  following  table: 

Examination  of  Urine. 


VOLTTME. 

ROTATION. 

IRISH  MOSS 

CC. 

-0.04° 



Injection 

128 

+  0.34° 

— 

226 

+  0.06° 

— 

330 

-0.20° 



370 

-0.14° 



May  18,  9  A.M. 
May  18,  4P.M. 
May  19,  9  A.M 
May  20,  9  A.M. 
May  21,  11A.M. 
May  22,     9  A.M 


Tests  for  Irish  moss  on  May  19th  were  negative,  but  on  May  20th- 
22nd  they  were  faintly  positive.  The  experiment  was  discontinued 
at  this  point.  The  injection  was  not  very  well  borne,  the  dog  remain- 
ing lethargic  throughout  the  period. 


2.  Intraperitoneal. 

Experiment  A .     A  dog  weighing  10  kg.  received  160  cc.  of  an  Irish 
moss  solution  containing  1.3  grams  air  dry  material.     Examination 


Nutrition  Investigations. 


337 


for  the  presence  of  carbohydrate  was  made  by  testing  the  urine  for 
reducing  substances,  before  and  after  hydrolysis.  The  results  are 
shown  in  the  following  table: 

Examination  of  Urine. 


REDUCTION    OF    FEHXING's 
SOLUTION. 


Before 
Hydrolysis. 


After  Hydroly- 


OctoberlS,  11  A.M 

October  13,  12:30  P.  M •. . .  .  Injection 

October  13,    2  P.M 27 

October  13,    5  P.M 60 

October  14,    9  A.M 450 

October  15,    5  P.M 45 

October  16,  9:30 A.M i  — 


The  urine  before  the  injection  showed  a  rotation  of  —0.14°,  a 
sample  of  the  mixed  urines  of  October  13,  5  P.M.,  and  October  14, 
9  A.M.,  showed  a  rotation  of  —0.034°,  the  diminished  levo-rota- 
tion  undoubtedly  due  to  the  presence  of  this  dextro-rotatory  carbohy- 
drate. On  hydrolysis,  50  cc.  of  this  mixed  sample  yielded  sugar 
equivalent  to  0.035  grams  of  dextrose  (by  Allihn's  method) .  From  the 
remainder  of  this  sample,  Irish  moss  carbohydrate  was  isolated;  it 
formed  a  grayish- white  powder,  sweUing  in  water,  and  yielding  mucic 
acid  on  oxidation  with  nitric  acid. 

Experiment  B.  A  dog  weighing  9  kg.  received  intraperitoneally 
100  cc.  of  a  2  per  cent  solution  of  Irish  moss  preparation.  Examina- 
tion for  carbohydrate  was  made  as  in  the  preceding  experiments. 
The  results  appear  in  the   following  table: 

Examination  of  Urine. 


REDUCTION  OF  FEHUNG 'S 
SOLUTION. 

[Before  Hydroly-  After  Hydroly- 

j             sis.                         sis. 

October  30     1  P.M     

cc. 

Injection               — 
250 

200                    — 
115                    — 

October  30,  2:30  P.M 

_ 

October  31,      9  A.M 

November  1,  10  A.M 

-I- 

November  2,  10  A.M 

Irish  moss  was  isolated  and  identified  in  the  urine  of  November  1st. 


338 


Mary  Davies  Swartz, 


INJECTIONS   OF   SALEP. 

I.  Subcutaneous. 

A  dog  weighing  7.2  kg.  received  56  cc.  of  salep  solution,  containing 
0.75  grams  of  pure  mannan.  No  reducing  substance  was  found  in 
the  urine.  The  changes  in  rotation,  due  to  salep,  are  shown  in  the 
following  table : 

Examination  of  Urine. 


TDSZ. 

VOLUME. 

ESTIMATION    OF    AMOUNT 
ROTATION. 

!      OF  SALEP  EXCRETED. 

May  17, 

CC. 

Injection 

138 
132 
114 

127 

-0.17° 

-  0.27° 
-0.27° 
-0.20° 
-0.14° 

Grams. 

May  18  330 P.M 

May  19,  9  A.M 

May  20  9  A.M 

0.3 

May  21  9  A.M 

0.3 

May22,  9  A.M 

0.04 

May  22  5  P.M 

Salep  was  isolated  and  identified  in  the  urines  of  May  20,  21,  and  22. 


2.  Intraperitoneal. 

Experiment  A .  A  dog  weighing  7  kg.  received  68  cc.  of  salep  solu- 
tion, containing  1.2  grams  of  air  dry  mannan.  No  reducing  substance 
was  present  in  the  urine  at  any  time.  Tests  for  the  presence  of  salep 
by  means  of  FeMing's  solution,  gave  the  following  results: 

Examination  of  Urine. 


TIME. 

VOLUME. 

SALEP  PRESENT. 

October  21,  12  M 

CC. 

Injection 
125 
190 

October  21  2-30  P.M 

October  22,  9  A.M 

+ 

October  23,  9  A.M 

4- 

October  24,  9  A.M 

140                               — 

The  salep  was  easily  isolated  and  identified  in  the  urine  of  October 
22  and  23,  the  sugar  obtained  on  hydrolysis  being  equivalent  to  0.33 
grams  salep. 

Experiment  B.     A  dog  weighing  9.2  kg.  received  80  cc.  of  salep  so- 


Nutrition  Investigations. 


339 


lution,  containing  1.4  grams  of  air  dry  substance.  No  reducing  sub- 
stance was  detected  in  any  of  the  urines.  Tests  for  salep  with  Feh- 
ling's  solution  gave  the  following  results: 

Examination  of  Urine. 

TIME.  '  VOLUME.  SAI^P  PRESENT. 


October  24,  11  A.M 

October  24,  12  M 

October  25, 12  M 155  + 

October  26, 10  A.M 180  + 

October  27, 10  A.M 180  + 

October  28, 10  A.M 


From  the  urine  of  October  25,  salep  was  isolated,  which  jdelded  on 
hydrolysis  0.39  grams  reducing  sugar  as  dextrose;  it  was  also  isolated 
from  the  urines  of  the  next  two  days,  but  was  not  estimated  quanti- 
tatively. 

Experiment  C.  A  dog  weighing  9.2  kg.  received  90  cc.  of  salep 
solution,  containing  1.8  grams  of  pure  mannan.  No  reduction  of 
Fehling's  solution  occurred  with  any  of  the  samples.  Tests  for  salep 
with  Fehling's  solution  gave  the  following  results: 


Examination  of  Urine. 


ROTATION. 


SALEP  PRESENT. 


December  2,  10  A.M i 

December  2,  2:30  P.M j  Injection 

December  3,  10  A.M i  960 

December  4, 10  A.M j  234 

December  5, 10  A.M I  520 


-0.17° 


-0.41° 
-0.27° 


+ 
+  (0.6gm.) 
+  (0.5gm.) 


Unfortunately  this  experiment  was  imavoidably  interrupted  at 
this  point.  The  salep  was  precipitated  from  50  cc.  of  the  urine  for 
December  3,  hydrolyzed,  and  sugar  determined  gra^dmetrically  as 
dextrose,  from  which  the  total  amount  of  salep  in  this  day's  urine  was 
calculated  as  0.67  gram.  Salep  determined  in  the  same  way  on  De- 
cember 4,  showed  an  elimination  of  0.18  gram;  hence  0.85  gram  was 
actually  recovered  in  these  two  days.  The  influence  of  the  levo- 
rotatory  carbohydrate  on  the  rotation  of  the  urine  was  marked. 


340 


Mary  Davies  Swartz 


Experiment  D.  A  dog  weighing  6.4  kg.  received  98  cc.  of  salep 
solution  containing  1  gram  of  pure  mannan.  No  reduction  of  Feh- 
ling's  solution  was  observed  throughout  the  experiment.  The  changes 
in  rotation  due  to  the  salep  are  shown  in  the  following  table: 


Examination  of  Urine. 


ROTATION. 


salep  precipitated  by 
fehling's  solution. 


January  31 
February  1 
February  2 
February  3 
Februar}'  4 
February  5 


116 
Injection 
152 
238 
154 
137 


-0.41° 

-0.41° 
-0.13° 
-0.13° 
-  0.20° 


+ 


The  results  in  this  experiment  are  very  puzzling.  The  normal  rota- 
tion was  high  (—0.41°)  for  several  weeks  before  this  experiment 
but  fairly  constant,  averaging  —0.44°.  If  salep  were  excreted  as 
mannan,  the  levo-rotation  should  have  increased,  yet  it  was  decid- 
edly low  on  a  day  when  salep  was  shown  to  be  present,  and  also  on  a 
day  when  none  could  be  detected.  The  absence  of  any  positive  tests 
for  sugar,  excluded  the  idea  that  the  salep  was  being  excreted  in  this 
form,  but  finally  a  sample  of  February  4,  was  tested  with  yeast,  and 
marked  fermentation  observed.  Unfortunately,  this  was  after  all  the 
other  samples  had  been  discarded,  hence  no  further  tests  could  be 
made. 

Experiment  E.  A  dog  weighting  9.8  kg.  received  intraperitoneally 
97.5  cc.  of  salep  solution  containing  1.3  grams  pure  mannan.  No  re- 
duction of  Fehling  's  solution  was  observed.  The  changes  in  rotation 
are  shown  in  the  first  table  on  the  next  page. 

Salep  was  isolated  and  identified  as  carbohydrate,  in  the  urines  of 
May  19,  20,  and  21,  although  the  amount  in  the  last  two  days  was  ap- 
parently too  small  to  be  detected  by  any  change  in  the  rotation. 


INJECTIONS   OE   SINISTRIN. 

I.  Subcutaneous. 

A  dog  weighing  6.5  kg.  received  49  cc.  of  sinistrin  solution,  contain- 
ing 3.3  grams  pure  substance.     This  solution  showed  a  rotation  of 


Nutrition  Investigations. 
Examination  of  Urine. 


341 


ESTIMATION    OF    AMOUNT 
01  SAIEP  EXCRETED. 


Mayl7, 10A.M 
May  18, 10  A.M 
May  18,  3  P.M 
May  19,  9  A.M 
May  20,    9  A.M 

May  21, 11  A.M 
May  22,    9  A.M 


Injection 
165 
250 


405 
200 


-0.14° 
-0.14° 

-0.34° 
-  0.14° 


-0.14° 
-0.14° 


Grams. 


0.4 

Salep  present — pre- 
cipitated by  Feh- 
ling's   Solution. 

Salep  present. 

No  Salep  present. 


-3.88°  in  a  200  mm.  tube.  The  urine  contained  no  reducing 
substance  at  any  time.  The  changes  in  rotation,  due  to  sinistrin  in- 
jection, are  shown  in  the  following  table: 

Examination  of  Urine. 


VOLUME. 


ESTIMATION  or  AMOUNT 
OB  SINISTRIN  EXCRETED.  * 


January  15, 12  M.... 
January  15,  2:30  P.M 

January  16 

January  17  and  18 . .  . 

January  19 

January  20 


Injection 

260 

165 

60 

108 


-0.41° 

-0.97° 
-0.41° 
-0.41° 
-0.47° 


Grams. 


2.5 


*  CalcTilatlng  f or  sinistrin  [a]  d  =  —29.1°. 


2.  Intraperitoneal. 

Experiment  A.  A  dog  weighmg  6.5  kg.  received  110  cc.  of  sinis- 
trin solution,  containing  2  grams  pure  substance.  This  solution 
showed  a  rotation  of  - 1.18°  in  a  200  mm.  tube.  No  reducing  sub- 
stance was  found  in  the  urmes  examined.  The  changes  m  rotation, 
due  to  sinistrin  injection,  are  shown  in  the  following  table: 


342 


Mary  Davies  Swartz, 
Examination  of  Urine. 


ROTATION. 


ESTIMATION  OF  AMOUNT 
OF  SINISTRXN  EXCRETED.* 


January  11,  10:30  A.M 

January  11,  3  P.M 

January  12,  9:30  A.M 

January  13,  9:30  A.M 

January  14,  9:30  A.M 

*  Calculating  for  slnlstrln  \a\  d 


Injection 

88 

127 

116 


-0.48° 

-  2.04° 

-  0.48° 
-0.48° 


Grams. 


2.7 


=  —29.1°. 

Experiment  B.  A  dog  weighing  4.6  kg.  received  108  cc.  of  sinis- 
trin  solution,  containing  2.3  grams  pure  substance.  The  rotation  of 
this  solution  was  —1.38"  in  a  200  mm.  tube.  No  reducing  sub- 
stance was  detected  in  the  urine  at  any  time.  The  changes  in  rota- 
tion are  shown  in  the  following  table: 

Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

ESTIMATION    OF    AMOUNT 
OF  SINISTRIN  EXCRETED.* 

January  26 

CC. 

Injection 

148 

95 

155 

-0.14° 

-  1.38° 
-0.41° 
-0.14° 

Grams. 

January  27,  9:30  A.M 

January  27,  5  :P.M 

January  28,  9  :AM 

2.1 
0.4 

January  29,  9  :A.M 

■  Calculating  for  slnlstrln  [a]  d  =  —  29.1° 


In  all  these  experiments,  the  sinistrin  was  isolated  and  identified  as 
a  levo-rotatory  carbohydrate,  yielding  reducing  sugar  on  hydrolysis. 
It  was  apparently  excreted  quantitatively  in  every  case. 


FEEDING  EXPERIMENTS. 

Methods  and   Technique. 

Feeding  experiments  were  conducted  with  dogs  and  human  sub- 
jects, under  conditions  as  nearly  normal  as  possible.  The  dogs  were 
kept  in  metal  cages,  arranged  for  the  separate  collection  of  urine  and 
faeces.  They  were  fed  once  a  day,  on  a  uniform  weight  diet,  consist- 
ing of  chopped  lean  meat,  lard,  and  cracker  meal,  in  suitable  portions 


Nutrition  Investigations.  343 

and  amounts  to  maintain  a  constant  body  weight.  The  carbohydrate 
under  investigation  was  dissolved  or  suspended  in  water,  and  mixed 
with  this  basal  ration.  In  the  earlier  experiments  the  periods  were 
divided  as  follows:  Fore  =  3  days  on  the  basal  ration;  mid  =  3  days 
in  which  some  preparation  was  added,  the  amoimt  being  the  same 
each  day;  after  =  3  days  like  the  fore  period.  Separation  of  the  pe- 
riods in  the  faeces  was  accomplished  by  marking  with  soot  or  carmine 
capsules.  In  all  later  experiments,  two  days  constituted  the  fore  pe- 
riod, and  a  day  on  the  normal  diet  was  included  at  the  beginning  and 
end  of  the  mid  period,  making  thus  four  days,  to  insure  against  any 
of  the  material  under  investigation  being  carried  into  the  faeces  of 
the  after  period. 

In  several  cases,  the  presence  or  absence  of  galactans  or  mannans 
in  the  faeces  has  been  verified  by  testing  the  hydrolyzed  material  for 
mucic  acid  or  mannose-hydrazone. 

For  analysis,  the  faeces,  collected  and  weighed,  were  rubbed  to  a 
thin  mud  with  alcohol,  dried  to  constant  weight  on  a  water  bath, 
weighed  air  dry,  and  ground  finely  in  a  coffee  mill.  The  portions 
constituting  each  period  were  thoroughly  mixed,  and  from  2  to  5 
grams  taken  for  hydrolysis,  according  to  the  yield  of  carbohydrate 
anticipated.  The  samples  were  boiled  on  a  reflex  condenser  with 
100  cc.  of  2  per  cent  hydrochloric  acid,  for  two  hours;  or  longer  if 
thought  to  contain  a  carbohydrate  which  previous  analysis^  had 
shown  to  require  more  time  for  complete  hydrolysis. 

The  products  of  hydrolysis,  cooled  and  neutralized,  were  made  up 
to  250  cc.  and  sugar  determined  as  dextrose  by  Allihn's  gravimetric 
method.  It  was  found  that  the  copper  reduction  was  often  very  in- 
complete, and  that  much  more  satisfactory  results  came  from  clari- 
fying the  solutions  with  charcoal  after  making  up  to  volume.  Not 
only  were  duplicate  analyses  in  closer  agreement,  but  in  some  cases 
the  yield  of  cupric  oxide  was  two  or  three  times  greater  than  before 
this  treatment.  Owing  to  the  complexity  and  diversity  of  the  prod- 
ucts of  hydrolysis,  results  are  at  best  only  approximate. 

In  experiments  with  dulse,  the  pentosans  were  determined  by  the 
phloroglucin  method.  ^ 

The  human  subjects  were  healthy,  active  young  women.  Their 
diet  was  not  weighed,  but  was  kept  as  uniform  as  possible.     All  cel- 


1  Cf.  table,  p.  317. 

*Cf.  Official  and  Provisional  Methods  of  Analysis,  Bulletin  No.  107  (1907), 
Bureau  of  Chemistry,  United  States  Department  of  Agriculture. 


344  Mary  Davies  Swartz, 

lulose-containing  foods,  such  as  nuts,  frmts,  green  vegetables,  peas 
and  beans,  coarse  bread  and  cereals,  were  carefully  avoided;  so  that 
the  carbohydrates  were  limited  almost  entirely  to  bread  and  crackers 
made  from  fine  white  flour,  a  small  quantity  of  potato,  and  sugar. 
To  this  diet  the  gelatinizing  carbohydrates  were  added  in  the  form  of 
blanc  mange  or  jelly;  dulse  was  dissolved  in  some  beverage,  and  the 
insoluble  preparations  boiled  half  an  hour  in  a  little  water  and  eaten 
as  a  vegetable,  seasoned  with  salt,  butter,  and  vinegar.  The  blanc 
manges  or  jellies  made  from  the  Hawaiian  seaweed  preparations  were 
equally  attractive  in  texture  and  flavor  with  those  made  from  Irish 
moss. 

Periods  were  marked,  and  the  analyses  of  faeces  conducted  in  the 
manner  already  described  for  the  experiments  with  dogs. 

The  Digestibility  of  Pentosans. 

Four  preparations  were  fed.  Dulse, ^  Limu  Eleele,^  Limu  Lipoa,^ 
and  Limu  Pahapaha,^  without  production  of  unpleasant  symptoms  in 
any  case.  The  results  of  all  trials  are  shown  in  the  tables  on  the 
following  pages. 


1  Cf.  p.  303. 

2  Cf.  p.  307. 

3  Cf.  p-  308. 


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348 


Mary  Davies  Swartz, 


The  coefScients  of  digestibility  of  the  pentosan  preparations,  as 
determined  in  the  usual  way  from  the  preceding  experiments,  are  set 
forth  in  the  following  table: 


SERIES  A. 

PENTOSAN. 

COEFFICIENT  OF 

DIGESTIBIUTY. 

EXPERIMENT  NO. 

For  the  Dog. 

For  Man. 

1 

Dulse 

80 

2 

Dulse 

66 

3 

Dulse 

100 

4 

Dulse 

100 

5 

Limu  Eleele 

50 

6 

Limu  Eleele 

20 

7 

Limu  Eleele 

69 

8 

Limu  Pahapaha 

34 

9 

Limu  Lipoa 

16 

It  is  evident  from  these  figures,  that  pentosans  in  soluble  form  dis- 
appear from  the  alimentary  tract  of  dogs  to  a  very  considerable  extent 
(average  73  per  cent),  and  that  small  quantities,  ingested  by  man,  do 
not  reappear  in  the  faeces.  The  insoluble  limu  preparations  appear 
much  more  indigestible,  an  average  of  28  per  cent  being  digested  by 
dogs,  and  51  per  cent  by  man. 

It  must  be  borne  in  mind,  in  interpreting  the  results  of  these  metabo- 
lism experiments,  that  they  are  at  best  only  approximate.  The  dif- 
ficulty of  strict  separation  of  the  faeces,  the  fact  that  the  human  sub- 
jects were  not  kept  on  a  uniform  weighed  diet,  and  the  errors  unavoid- 
ably introduced  by  determining  many  different  kinds  of  sugar  as  dex- 
trose, make  all  of  the  figures  given  as  "coefiicients  of  digestibility," 
in  this  and  succeeding  sections,  comparative  rather  than  absolute. 


The  Digestibility  of  Galactans. 

In  these  experiments,  preparations  of  the  water  extracts  of  Irish 
moss,  Limu  Manauea,  Limu  Huna  and  Limu  Akiaki  have  been  fed, 
without  any  disagreeable  symptoms.  The  results  are  given  in  the 
tables  which  follow: 


Q 

W 

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352 


Mary  Davies  Swartz, 


The  coefficients  of  digestibility  of  the  galactan  preparations  are  given 
in  the  following  table: 


SERIES   B. 

GALACTAN. 

COEFFICIENT  OI 

DIGESTIBILITY. 

EXPERIMENT   NO. 

For  the  Dog. 

For  Man. 

Per  cent. 

Per  cent. 

1 

Irish  Moss 

46 

2 

Irish  Moss 

20 

3 

Irish  Moss 

11 

4 

Irish  Moss 

0 

5 

Limu  Manauea 

55 

6 

Limu  Manauea 

12 

7 

Limu  Manauea 

30 

8 

Limu  Manauea 

30  (av.) 

9 

Limu  Huna 

30  (20  gms. 
fed) 

10 

Limu  Huna 

83  (7  gms.- 
fed) 

11 

Limu  Huna 

10 

12 

Limu  Akiaki 

60 

Although  these  preparations  were  administered  in  small  quanti- 
ties, under  the  most  favorable  conditions  for  digestion  in,  the  only 
instance  where  the  utilization  in  any  degree  approaches  that  of  starch 
(Limu  Huna),  the  quantity  fed  (7  grams)  was  so  small  that  this  exper- 
iment can  hardly  be  taken  as  a  criterion  of  digestibility.  Exclusive 
of  this  experiment,  the  average  of  five  trials  with  dogs  is  32  per  cent, 
while  that  of  six  trials  with  human  subjects  is  23  per  cent.  In  both 
cases,  the  averages  are  lower  than  that  of  Lohrisch  (194)  for  ''soluble 
agar,"  50  per  cent. 

Where  the  quantity  of  galactan  fed  was  10  or  more  grams,  the  in- 
fluence on  the  character  of  the  faeces  was  usually  noticeable.  The 
increase  in  bulk,  after  ingestion  of  45  grams  of  Irish  moss,  is  well  illus- 
trated in  a  photograph  of  the  dried  and  groimd  faeces  of  the  dogs 
used  in  experiments  1  and  2:^ 

A  represents  the  fore-period  (3  days),  B  the  mid-period,  during  which 
15  grams  of  moss  were  ingested  daily  (3  days),  and  C  the  after-period 
(3  days).  The  separation  of  the  faeces  at  the  beginning  of  experi- 
ment 1  (on  the  right)  was  not  very  satisfactory.  The  dog  had  pre- 
viously been  fed  bone-ash,  and  the  marked  faeces  were  undoubtedly 
contaminated  with  this,  so  that  they  appear  unusually  bulky.  Exper- 
iment 2  is  typical  of  the  results  obtained  in  most  of  the  experiments 


1  Cf.  p.  343. 


Nutrition  Investigations. 


353 


with  human  subjects.  In  these,  the  undigested  hemicelluloses  gave 
frequently  a  pecuhar,  wax-hke  consistency,  especially  noticeable  with 
Limu  Huna  in  the  experiment  recorded,^  and  in  another  not  reported, 
because  the  faeces  for  part  of  the  time  were  lost.  In  the  experiment 
with  Limu  Akiaki  (No.  12), ^  the  galactan  was  excreted  after  the  first 
day's  feeding,  in  a  tough  mass  almost  impossible  to  break  up  with  a 


EXP.   2 


EXP.    1 


I. 


The  Influence  of  Irish  Moss  upon  the  Mass  of  the  Faeces. 

A.  Fore  Period:     3  Days  on  a  CeUulose-free  Diet. 

B.  Mid  Period:     3  Days  on  a  Cellulose-free  Diet  to   Which   15  grams  of 

Irish  Moss  were  Added  Daily. 

C.  After  Period:   3  Days  of  a  Cellulose-free  Diet. 

spatula.  That  of  the  second  day  was  not  excreted  till  the  third 
day  after  feeding,  the  subject  being  inclined  to  constipation.  It  seems 
likely  that  the  high  coefiicient  of  digestibility  is  due  to  this  fact,  or 
else  to  the  method  of  determination,  which  is  not  altogether  satisfac- 
tory, in  view  of  the  complexity  of  the  products  of  hydrolysis,  the  dan- 
ger of  decomposing  a  part  of  the  sugar  from  the  easily  inverted 
polysaccharides  by  the  long  boiling  necessary  for  the  more  resistant, 
and  the  great  difference  in  reducing  power  of  the  sugars  so  produced. 

The  Digestibility  of  Mannan. 

In  four  experiments,  the  commercial  salep  powder  (containing  19 
per  cent  mannan  and  26  per  cent  starch)  was  administered;  in  the 
others,  pure  mannan  prepared  from  the  Orchis  tubers.  The  results 
of  seven  trials  are  tabulated  on  the  following  pages. 

1  Cf.  p.  345. 


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356 


Mary  Davies  Swartz. 


The  coefi&cients  of  digestibility  of  the  salep  preparations  are  shown 
in  the  following  table : 


SERIES   C. 
EXPERIMENT  NO. 


COEFFICIENT  OF  DIGESTIBrLlTY. 


For  the  Dog. 


For  Man. 


Per  cent 

Per  cent 

1 

Salep  Powder 

70 

2 

Salep  Powder 

100 

3 

Salep  Powder 

100 

4 

Salep  Powder 

94 

5 

Salep  Mannan 

10 

6 

100 

7 

100 

Thus  we  see  that  in  every  case,  except  that  in  which  a  dog  received, 
in  one  day,  10  grams  of  pure  mannan,  the  greater  portion  of  the  salep 
fed  was  digested,  the  coefi&cient  of  salep  powder  for  dogs  averaging 
85  per  cent,  and  for  man,  97  per  cent;  while  that  of  pure  mannan  for 
man  is  100  per  cent,  in  spite  of  the  fact  that  it  is  not  attacked  by  diges- 
tive enzymes! 

The  contrast  between  the  volume  of  faeces  produced  when  a  galac- 
tan  such  as  Irish  moss  was  fed,  and  that  when  a  more  digestible  hemi- 
cellulose  was  given,  is  shown  in  the  photograph  of  the  faeces  from 
experiments  Nos.  1  and  2  of  Series  C,^  on  the  next  page,  in  which  A 
represents  the  fore-period,  B  the  mid-period,  and  C  the  after-period, 
each  period  being  three  days  in  duration.  The  group  on  the  right 
represents  experiment  No.  1,  in  which  70  per  cent  of  the  hemicellulose 
and  starch  of  the  salep  powder  was  digested,  and  that  on  the  left, 
experiment  No.  2  in  which  apparently  all  of  these  were  digested. 


DISCUSSION  AND   SUMMARY. 

A  glance  at  the  table  on  page  327  clearly  shows  that  none  of  the 
hemicelluloses  tmder  consideration  are  readily  attacked  by  the  ordi- 
nary animal  or  vegetable  enzymes.  The  results  are  for  the  most  part 
entirely  negative.  Even  where  there  has  been  hydrolysis  with  0.2 
per  cent  hydrochloric  acid,  the  amount  of  sugar  produced  in  24  hours 
was  relatively  small.  The  hydrolyzing  action  of  the  gastric  juice 
is  probably  largely  due  to  the  presence  of  acid,  although  no  compari- 
son of  the  relative  amoimts  of  sugar  produced  by  gastric  juice  or  by 

^Cf.  p.  348. 


Nutrition  Investigations. 


357 


0.2  per  cent  acid  alone  has  been  made.  It  is  noticeable  that  even  the 
very  soluble  hemicellulose,  sinistrin,  which  is  so  speedily  hydrolyzed 
by  acid  (in  |  hour  at  37°  C.  with  0.2  %  hydrochloric  acid)  is  not  attacked 
by  ordinary  diastatic  enzymes  within  24  hours. 

The  parenteral  introduction  of  these  carbohydrates  has  resulted  in 
their  speedy  and  apparently  complete  eHmination  through  the  kid- 
neys without  any  change  in  character.  The  carbohydrates  prepared 
from  Dulse,  Irish  Moss,  Salep  and  Sinistrin  have  all  been  isolated  and 
identified  in  the  urine,  after  subcutaneous  and  intraperitoneal  injec- 
tions.    These  results  are  not  surprising,  in  view  of  the  commonly  ac- 


EXP.   1 


IT. 


The  Influence  of  Salep  upon  the  Mass  of  the  Faeces. 

A.  Fore  Period:    3  Days  on  a  Cellulose-free  Diet. 

B.  Mid  Period:     3  Days  on  a  Cellulose-free  Diet  to  Which  15  grams  of 

Salep  Powder  were  Added  Daily. 

C.  After  Period:    3  Days  on  a  Cellulose-free  Diet. 

cepted  fact  that  carbohydrates  must  be  converted  into  monosacchar- 
ides before  they  can  enter  into  the  processes  of  intermediary  meta- 
bolism. 

Experimental  evidence  in  support  of  this  fact  is  given  by  such 
investigators  as  F.  Voiti  and  Blumenthal,^  who  found  that  even  di- 
saccharides,  as  lactose  and  saccharose,  were  eliminated  almost  quanti- 


^Miinchener  medicinische  Wochenschrift,  1896,  p.  717;  Deutsches  Archiv  fiir 
klinische  Medicin,  v.  58,  p.521  (1897). 

^  Beitage  zur  chemischen  Physiologie,  v.  6,  p.  329  (1905) . 


358 


Mary  Davies  Swariz, 


tatively  after  subcutaneous  injection  in  man  and  the  rabbit;  or  as 
Mendel  and  Mitchellj^  who  have  shown  that  polysaccharides  like 
dextrin,  soluble  starch,  glycogen,  inulin,  and  isolichenin  are  recovered 
to  a  considerable  extent  in  the  urine,  whether  introduced  subcuta- 
neously,  intraperitoneally,  or  intravenously. 

In  the  present  experiments,  the  dulse  pentosan  was  the  most  slowly 
eliminated,  being  found  in  the  urine  four  or  five  days  after  injection; 
Irish  moss  and  salep  were  not  detected  after  the  third  day;  while  si- 
nistrin  seemed  to  be  all  excreted  within  the  first  24  hours. 

The  average  coefi&cients  of  digestibility  for  the  ten  preparations 
which  have  formed  the  basis  of  the  feeding  experiments,  are  summar- 
ized in  the  following  table : 

Coefficients  of  Digestibility  of  Hemicelluloses. 


HEMICELLITLOSE. 

SUBJECT   OF  EXPERIMENT. 

Class. 

Source. 

Dog. 

Man. 

Pentosan 

Dulse 

Limu  Eleele 
Limu  Pahapaha 
Limu  Lipoa 
Irish  Moss 
Limu  Manauea 
Limu  Huna 
Limu  Akiaki 
Salep  Powder 
Salep  Mannan 

Per  cent. 
73  (2  exp.) 
35  (2  exp.) 

16  (1  exp.) 
33  (2  exp.) 
33  (2  exp.) 
56  (2  exp.) 

85  (2  exp.) 
10  (1  exp.) 

Per  cent. 
100  (2  exp.) 
9  (2  exp.) 
34  (1  exp.) 

Pentosan 

Pentosan 

Pentosan 

Galactan 

Galactan 

Galactan 

6  (2  exp.) 
30  (3  exp.) 
10  (1  exp.) 
60(1  exp.)* 
97  (2  exp.) 
100  (2  exp.) 

Galactan 

Mannan . 

*  Subject  with  chronic  constipation. 

That  the  low  coefi&cients  enumerated  above  are  not  due  to  inabil- 
ity of  the  various  subjects  to  utilize  carbohydrates,  is  shown  by  the 
following  figures. 

The  coefficient  of  digestibility  for  cracker  meal  in  the  experiments 
on  dogs,  determined  by  taking  the  average  of  all  the  fore-periods  of 
the  feeding  trials,  in  which  five  different  dogs  were  used,  was  99.0 
per  cent.  This  is  much  higher  than  London  and  Polowzowa's^  coef- 
ficient for  carbohydrate  digestibility  in  dogs  on  a  bread  diet,  96  per 
cent. 


1  American  Journal  of  Physiology,  v.  14,  p.  239  (1905). 
^Zeitschrift  fur  physiologische  Chemie,  56,  513  (1908). 


Nutrition  Investigations.  359 

For  the  four  women  who  were  subjects  of  feeding  experiments,  the 
average  daily  amount  of  carbohydrate  excreted  in  the  faeces,  on  a 
cellulose-free  diet,  estimated  as  dextrose,  by  averaging  the  fore-pe- 
riods of  all  trials,  was  0.8  gram.  The  utilization  of  carbohydrates  was 
therefore  unusually  good,  since  Atwater  and  Bryant's^  coefi&cient  of 
digestibility  for  such  a  diet  is  98  per  cent,  and  undoubtedly  every  one 
of  these  individuals  consumed  over  50  grams  of  carbohydrate  per  day. 
With  the  exception  of  the  subject  of  a  single  experiment  who  had 
chronic  constipation,  these  were  all  normal,  healthy  individuals,  free 
from  disturbances  of  the  alimentary  tract. 

The  three  seaweeds  fed  in  toto,  Limu  Eleele,  Limu  Pahapaha,  and 
Limu  Lipoa,  show  an  average  digestibility  of  51  per  cent.  This  is 
higher  than  that  obtained  in  Professor  Mendel's  laboratory  for  un- 
cooked Cetraria  islandica^  (average  of  three  experiments,  15  per  cent) 
and  much  lower  than  that  reported  by  Oshima  for  dried  marine  algae' 
(average  77  per  cent). 

In  man,  with  the  exception  of  dulse  and  salep,  which  almost  entirely 
disappeared  in  the  alimentary  tract,  the  average  digestibility  of  all 
preparations  is  only  34  per  cent,  a  figure  in  contrast  to  those  of  Loh- 
risch  (194),  who  finds  cellulose  and  hemicellulose  50  per  cent  digestible. 
In  dogs,  the  average  of  all  preparations  is  42  per  cent. 

Considering  that  the  pentosan  of  dulse  was  in  a  form  most  favorable 
for  digestion,  the  results  with  this  hemicellulose  are  in  harmony 
with  those  of  Konig  and  Reinhardt  (120)  who  reported  75  per  cent  of 
the  pentosans  as  disappearing  from  the  aHmentary  tract  in  man; 
and  with  the  averages  obtained  by  the  various  investigators  on  ani- 
mals, which  show  these  carbohydrates  40-70  per  cent  diges- 
tible in  herbivora.*  It  would  be  desirable  to  repeat  the  experiments 
with  larger  quantities,  although  the  process  of  preparing  the  material 
is  rather  laborious.  It  must  be  borne  in  mind,  that  the  dulse  pento- 
san is  not  attacked  by  ordinary  diastatic  enzymes,  but  can  be  decom- 
posed by  soil  and  faecal  bacteria;  and  although  this  decomposition 
did  not  occur  readily  in  pure  solutions  of  the  carbohydrate,  or  even 
in  a  putrefying  mixture,  it  still  remains  to  be  demonstrated  whether 
the  complete  disappearance  from  the  alimentary  tract  is  not  largely 
due  to  the  more  favorable  conditions  for  bacterial  activity  within  the 


1  Report  Storr's  Agricultural  Experiment  Station,  1899,  p. 

2  Cf.  pp.  297-298. 

3  Cf.  p.  299. 

*  Cf.  pp.  274^275. 


360  Mary  Davies  Swariz, 

organism.  While  we  have,  in  the  case  of  herbivora,  some  convincing 
evidence  that  the  pentosans  are  a  true  source  of  energy, ^  we  have  as 
yet  no  real  grounds  for  this  assumption  in  the  case  of  man. 

The  insoluble  pentosans  of  the  Hawaiian  algae  are  manifestly  less 
digestible  than  the  soluble  forms.  The  coefi&cient  of  digestibility  is 
approximately  the  same  as  Slowtzoff' s  (154)  average  for  pure  xylan 
in  rabbits,  55  per  cent.  While  it  would  be  perhaps  desirable  to  de- 
termine the  pentosans  directly  by  the  furfurol-phloroglucin  method, 
rather  than  by  estimation  of  sugar  after  acid  hydrolysis,  a  trial  with 
dulse  by  both  methods  gave  practically  identical  results:  hence,  con- 
sidering that  the  hemicelluloses  of  these  algae  are  chiefly  pentosans, 
it  seems  safe  to  assume  that  the  results  reported  represent  the  amount 
of  pentosan  excreted,  within  the  limits  of  error  for  all  of  the  feeding 
experiments. 

The  galactans  were  all  soluble,  and  were  ingested  in  quantities  not 
exceeding  15  grams  per  day,  yet  the  coefi&cient  of  digestibility  is  lower 
than  for  any  other  hemicelliilose  group  (26  per  cent) .  The  resistance 
of  Irish  moss  is  particularly  striking,  but  is  not  surprising  in  view  of 
its  utter  indifference  to  attacks  of  digestive  enzymes  or  bacteria.  Its 
influence  on  the  character  of  the  faeces  was  not  so  marked  as  that  of 
Limu  Hima,  owing  probably  to  a  greater  tendency  to  liquefy  at  body 
temperature.  The  latter  would  seem  to  be  a  very  effective  agent  in 
constipation;  a  comparison  of  its  efficiency  with  that  of  agar-agar 
would  be  extremely  interesting.  Saiki  (205)  found  the  coefi&cient  of 
digestibility  for  agar  (average  of  two  experiments)  17  per  cent. 

In  view  of  the  negative  results  of  digestions  in  vitro  and  of  trials 
with  bacteria,  we  can  scarcely  be  surprised  at  the  results  of  these  met- 
abolism experiments,  especially  as  we  recall  that  Lohrisch  (57)  found 
that  his  "soluble  agar,"  already  partially  hydrolyzed,  was  only  diges- 
tible to  50  per  cent  (average). 

The  mannans  stand  in  striking  contrast  to  the  galactans.  In  the 
present  studies,  99  per  cent  of  the  salep  administered  has  been  uti- 
lized, a  result  in  accordance  with  Kano  and  lishima's  (255)  coefi&cient 
of  digestibility  for  the  Japanese  mannan,  Konjaku,  82  per  cent. 
Pure  mannan  fed  to  a  dog,  was  excreted  the  succeeding  day,  seem- 
ingly unaltered,  since  it  formed  a  semi-transparent  gelatinous  mass  in 
the  faeces,  from  which,  later,  a  rich  yield  of  mannose-hydrazone  was 
obtained.  The  very  different  result  with  salep  powder,  of  which  85 
per  cent  was  digested  by  dogs,  may  perhaps  be  accoimted  for  by  the 

^  Cf.  KeUner,  p.  274. 


Nutrition  Investigations.  361 

fact  that  it  contained  a  high  percentage  of  starch  (26  per  cent).  The 
amount  of  undigested  carbohydrate  excreted  in  the  faeces  is  in  close 
agreement  with  the  quantity  of  pure  mannan  ingested.  However, 
as  tests  for  mannose-hydrazone  were  negative  in  these  cases,  further 
experiments  are  necessary  before  an  authoritative  statement  can  be 
made  in  regard  to  this  question. 

It  is  manifestly  possible  for  faecal  and  soil  bacteria  to  produce 
sugar  from  mannan  ;i  hence  it  is  not  imlikely  that  hemicelluloses  of 
this  group  are  inverted  in  the  intestines  through  the  activity  of  micro- 
organisms, and  that  the  sugar  so  produced  is  absorbed  and  becomes  a 
true  source  of  energy  for  man,  in  spite  of  the  resistance  of  mannans  to 
the  action  of  digestion  enzymes.  Further  investigations  to  determine 
its  exact  nutritive  value  seem  highly  desirable. 

In  considering  the  proper  place  in  the  dietary  for  marine  algae, 
lichens  and  similar  substances,  we  must  not  disregard  the  possibility 
of  their  having  a  valuable  function  entirely  aside  from  the  question 
of  energy  production.  As  Oshima  (15)  points  out,  they  may  be  val- 
uable for  their  inorganic  salts.  The  non-irritating,  laxative  proper- 
ties of  many  species  make  them  desirable  adjuncts  to  the  diet  of  per- 
sons with  a  tendency  to  constipation;-  and  even  if  they  disappear,  in 
marked  degree,  from  the  alimentary  tract  during  the  process  of  diges- 
tion, they  may  perhaps  still  play  an  important  role  as  stimulants  to 
intestinal  activity,  being  in  fact  what  Prausnitz^  calls  "faeces-forming 
foods."  An  illustration  of  this  effect  is  afforded  by  the  experiments 
in  which  salep  powder  was  fed  to  dogs.*  The  periods  were  equal  in 
length,  and  in  one  case  (No.  2  in  photograph)  the  utilization  of  carbo- 
hydrates was  equally  good  for  all  three;  yet  in  the  mid-period  there 
is  a  decided  increase  in  the  bulk  and  weight^  of  the  faeces,  not  more 
than  1  gram  of  which  is  by  any  possibility  attributable  to  the  cellu- 
lose of  the  salep  powder,  and  in  the  other  experiment,  the  increased 
amount  of  faeces  cannot  be  wholly  accounted  for  by  the  amount  of 
undigested  carbohydrate  present. 

Mendel  (196)  has  already  sounded  a  warning  against  the  hasty 
assumption  that  every  carbohydrate,  by  virtue  of  its  ultimate  chem- 
ical composition,  stands  in  the  category  of  true  nutrients  for  the  human 
organism.     The  results  of  the  present  investigations  emphasize  the 


1  Cf .  Sawamura  (267) . 

2  Cf.  p.  283. 

^Zeitschrift  fiir  Biologic,  v.  35,  p.  335  a897). 

4  Cf.  pp.  348-349. 

5  Cf.  Table,  p.  348,  Series  C,  Experiments  Nos.  1  and  2. 


362  Mary  Davies  Swartz, 

necessity  of  drawing  our  final  conclusions  only  from  exact  metabolism 
experiments.  The  soluble  hemicelluloses  show  great  diversity  of 
behavior  in  the  alimentary  tract,  although  equally  resistant  to  diges- 
tive enzymes  in  vitro;  some  disappear  entirely,  others  reappear  in  the 
faeces  in  varying  degree,  up  to  100  per  cent.  It  is  evident  that  the 
latter  do  not  constitute  a  source  of  energy  for  the  organism:  how  far 
the  former  actually  do  so,  remains  to  be  demonstrated. 


IV.     CONCLUSIONS. 

1.  The  hemicelluloses  of  the  ten  species  of  marine  algae  included 
in  these  investigations  are  chiefly  pentosans  and  galactans.  The  pen- 
tosans are  largely  insoluble  in  water,  but  a  soluble  form  in  consider- 
able quantity  has  been  isolated  from  Rhodymenia  palmata.  The  ga- 
lactans are  soluble  in  hot  water,  and  are  characterized  by  their  gela- 
tinous nature.  Small  quantities  of  soluble  pentosans  have  been  found 
associated  with  them  in  every  case. 

2.  In  order  of  resistance  to  the  action  of  bacteria,  the  hemicellu- 
lose  groups  studied  stand  as  follows,  —  galactans,  pentosans,  levu- 
lans,  mannans,  the  galactan  of  Chondrus  crispus  being  entirely  unaf- 
fected by  common  micro-organisms. 

3.  Aerobic  and  anaerobic  cultures  of  soil  and  faecal  bacteria,  and 
cultures  of  B.  anthracis  symptomatici  and  B.  maligni  oedematis,  caused 
inversion  of  salep  mannan,  with  actual  production  of  reducing  sugar. 
The  latter  cultures  also  hydrolyzed  the  pentosan  of  Rhodymenia  pal- 
mata, and  the  levulan,  sinistrin.  In  a  mixture  of  aerobes,  salep  ap- 
peared to  be  partially  hydrolyzed,  forming  an  insoluble  transition 
product. 

4.  Digestion  experiments  in  vitro,  continued  for  24  hours  at  body 
temperature  under  antiseptic  conditions,  have  been  almost  entirely 
negative  in  result.  The  only  exceptions  are  the  hydrolysis  of  the  pento- 
san of  dvilse,  the  galactan  of  limu  kohu,  and  the  levulan,  sinistrin, 
by  "Taka"  diastase;  and  of  sinistrin,  and  the  galactans  of  hmu  kohu, 
limu  akiaki,  and  slippery  elm  bark,  by  artificial  gastric  juice  or  0.2 
per  cent  hydrochloric  acid,  the  action  of  the  gastric  juice  being  in  all 
probability  due  to  its  acidity. 

5.  After  parenteral  injection,  whether  subcutaneous  or  intra- 
peritoneal, the  hemicelluloses  are  excreted  through  the  kidneys,  and 
can  be  recovered  unaltered  in  the  urine.  The  pentosan  of  dulse  is 
completely  eliminated  in  four  to  five  days,  and  the  carbohydrates  of 
Irish  moss,  salep  and  sinistrin,  in  one  to  three  days. 

6.  Feeding  experiments  show  that  those  hemicelluloses  most 
readily  attacked  by  bacteria  disappear  most  completely  from  the 
alimentary  tract.  The  average  coefficient  of  digestibiHty  for  man  is, 
in  the  case  of  the  pentosan  of  dulse  and  the  mannan  of  salep,  99  per 

363 


364  Mary  Davies  Swartz, 

cent  notwithstanding  their  apparent  resistance  to  amylolytic  enzymes 
and  the  hydrolyzing  influence  of  the  gastric  jiiice;  their  disappear- 
ance seems  therefore  directly  attributable  to  bacterial  activity,  and 
the  possibility  of  sugar  formation  by  this  agency  having  been  demon- 
strated, it  remains  to  be  shown  by  means  of  respiration  experiments 
to  what  extent  materials  so  hydrolyzed  can  serve  as  true  nutrients  for 
the  organism.  Dogs  can  also  utilize  the  dulse  pentosan  to  a  consider- 
able degree,  but  their  power  to  digest  mannan  is  still  an  open  question. 
In  striking  contrast  to  the  above  hemicelluloses  stand  the  galac- 
tans,  with  their  high  degree  of  resistance  to  bacterial  decomposition; 
they  show  in  man,  an  average  digestibility  of  approximately  25  per 
cent,  in  dogs  of  45  per  cent.  It  is  manifestly  impossible  to  treat  of 
the  digestibility  of  hemicelluloses  as  a  class,  in  view  of  such  diversity 
in  the  groups.  Not  only  must  each  type  receive  special  considera- 
tion, but  distinction  must  be  drawn  between  soluble  and  insoluble  forms, 
as  is  illustrated  by  the  pentosans,  the  ratio  of  the  digestibility  coeffi- 
cient of  the  former  tothe  latter  being  approximately  100  to  50  in  man, 
and  75  to  25  in  dogs.  We  may,  however,  say  in  general,  that  they  disap- 
pear from  the  alimentary  tract  of  men  and  animals  to  an  extent  seem- 
ingly proportional  to  their  susceptibility  to  attacks  of  micro-organ- 
isms, and  give  little  justification  for  any  high  claims  made  for  them  as 
sources  of  energy  in  nutrition.  They  may,  however,  have  a  valuable 
function  as  adjuvants  in  the  dietary,  as  therapeutic  agents  in  consti- 
pation, or  as  sources  of  inorganic  salts. 

The  author  gratefully  acknowledges  the  helpful  suggestions  and 
criticism  freely  given  by  Professor  Lafayette  Mendel  throughout  the 
progress  of  this  work  and  the  kindly  interest  and  assistance  of  Pro- 
fessor Rettger  in  the  bacteriological  problems. 


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(103)  Cremer:  tJber  die  Verwertung  der  Rhamnose  im  tierischen  Organismus, 
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Nutrition  Investigations.  371 

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(118)  Kellner  and  Kohler:  Untersuchungen  iiber  den  Stoff- und  Energie- 
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(124)  Lindsey  and  Holland:  Twelfth  Massachusetts  State  Report,  p.  175, 
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(125)  Lintner  and  Dull:  Uber  die  chemische  Natur  des  Gerstengummi. 
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(130)  Pacault:  Sur  deux  proprietes  diastatiques  de  la  salive  de  I'escargot. 
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(132)  Reichardt:  Pararabin,  ein  neues  Kohlehydrat.  Berichte  der  deut- 
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(133)  Reinhardt:  Die  Bestimmung  der  Cellulose  und  ihr  Verhalten,  sowie 
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372  Mary  Davies  Swartz, 

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(140)  Salkowski:  Ueber  die  Darstellung  des  Xylans.  Ibid.,  V.  34,  p.  162, 
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(141)  Scheibler:  tJber  den  Pectinzucker  (Pectinose),  eine  neue  durch  Spal- 
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(149)  SelliJire:  Sur  une  diastase  hydolysant  la  xylane  dans  le  tube  digestif 
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(150)  Selliere:  Sur  la  presence  de  la  xylanase  chez  differents  moUusques 
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(151)  Selliere:  Sur  I'absorption  et  la  presence  dans  le  sang  chez  I'escargot 
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(152)  Selliere:  Sur  la  digestion  de  la  xylane  chez  quelques  mammiferes 
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(156)  Stone  and  Jones:  Verdaulichkeit  der  Pentosane.  Centralblatt  fiir 
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(157)  ToLLENS:  tJber  die  in  den  Pflanzenstoffen  und  besonders  den  Futter- 
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(158)  ToLLENS:  Untersuchungen  iiber  Kohlenhydrate.  Die  Landwirtschaft- 
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(159)  ToLLENS  AND  Glaubiiz:  tjber  den  Pentosan-gehalt  verschiedener 
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Nutrition  Investigations.  373 

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(160)  Tromp  De  Haas  and  Tollens:  Untersuchungen  iiber  die  Pectionstoffe. 
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(161)  Utzjanzew:  Zur  Physiologie  des  Blinddarmes  bei  den  Pflanzenfressem. 
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(167)  Wlnterstein:  tJber  das  pflanzliche  Amyloid.  Zeitschrift  fiir  physiolo- 
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(172)  Bente:  Zur  DarsteUung  der  Levulinsaine  und  iiber  Carragheenzucker. 
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(175j  Brasch:  Ueber  das  Verhalten  nicht-garungsfahiger  Kohlehydrate  im 
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(176)  Castoro:  Beitrage  zur  Kenntnis  der  HemiceUulosen.  Zeitschrift  fiir 
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374  Alary  Davies  Swartz, 

(178)  Fluckixger  and  Mayer:  Neues  Repertorium  fiir  Pharmacie,  1868, 
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(179)  Goret:  Etude  chimique  et  physiologique  de  quelques  albumens  comes 
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(181)  Greenish:  Die  Kohlehydrate  des  Fucus  Amylaceous.  Ibid.,  V.  5, 
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(182)  Gran:  Die  Hydrolyse  des  Agars  durch  ein  Enzym.  Centralblatt  fiir 
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(183)  Gruss:  Studien  uber  Reservecellulose.  Botanisches  Centralblatt,  V. 
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(184)  Gruss :  Ueber  den  Umsatz  bei  der  Keimung  der  Dattel.  Berichte  der 
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(186)  Herissey:  Isolement  du  galactose  crystallise  dans  les  produits  de 
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(187)  Herissey':  Recherches  chimiques  et  physiologiques  sur  la  digestion 
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(188)  HoFiiEiSTER:  Ueber  Resorption  imd  Assimilation  der  Nahrstoffe. 
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(190)  KoNiG  AND  Bettels:  Die  Kohlenhydrate  der  Meeresalgen  und  daraus 
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(191)  Lindsey:  The  Digestibihty  of  Galactan.  17th  Annual  Report,  Massa- 
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(194)  Lohrisch:  Der  Vorgang  der  Celliilose-  und  Hemicellulosenverdauung 
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(195)  Mallevre:  Der  Einfluss  der  als  Gahrungsprodukt  der  Cellulose  gebil- 
deten  Essigsaure  auf  dem  Gaswechsel.  Pfliiger's  Archiv  fiir  Physiologic,  V.  49, 
p.  460,   (1891). 

(196)  IMendel:  Das  Verhalten  einiger  unverdaulicher  Kohlehydrate  im  Ver- 
dauungstrakt.  Zentralblatt  fiir  die  gesammtc  Physiologic  und  Pathologie  des 
Stoffwechsels,  No.  17,  p.  1,  (1908). 

(197)  MtJLLER,  ELiRL:  Die  chemische  Zusammensetzung  der  Zellmembranen 
bei  verschiedenen  Kryptogamen.  Zeitschrift  fiir  physiologische  Chemie,  V.  45, 
p.  264,   (1905). 


Nutrition  Investigations.  375 

(198)  Munk:  Der  Einfluss  des  Glycerins,  der  fllichtigen  und  festen  Fettsauren 
auf  den  Gaswechsel.     Pfluger's  Archiv,  V.  46,  p.  303,  (1890). 

(199)  MtJNTZ:  Siir  la  Galactine.     Comptes   Rendus,   V.  94,  p.  453,  (1882). 

(200)  ]\Iuther:  Untersuchungen  iiber  Fucusarten.  Laminaria  und  Carraghe- 
enmoos,  sowie  die  hydrolytisch  daraus  entsehenden  Substanzen  und  iiber  Derivate 
derselben,  besonders  Fucose  und  Fuconsaure.  Inaugural  Dissertation,  Gottingen, 
1903. 

(201)  MtJTHEE.  AND  ToLLENs:  Ueber  die  Producte  der  Hydrolyse  von  Seetang 
(Fucus)  Laminaria,  und  Carragheen-Moos.  Berichte  der  deutschen  chemischen 
GeseUschaft,  V.  37,  p.  298,  (1904). 

(202)  Payen:  Sur  la  gelose  et  les  nids  de  salagane.  Comptes  Rendus,  V. 
49,  p.  521,  (1859). 

(203)  Pletnew:  Vergleichende  Ausnutzungsversuche  an  normalen  und  habi- 
tuell  obstipirten  Menschen.  Zeitschrift  fiir  experimentelle  Pathologie  imd  Thera- 
pie,  V.  5,  p.  186,  (1908). 

(204)  RoTHENEUSSER :  Der  Schleimkorper  des  Leinsamens.  Jahresbericht  fiir 
Thierchemie,  V.  34,  p.  78,  (1904). 

(205)  Saiki:  The  Digestibihty  and  Utilization  of  some  Polysaccharide  Carbo- 
hydrates derived  from  Lichens  and  Marine  Algae.  The  Journal  of  Biological 
Chemistry,  V.  2,  p.  251,  (1906). 

(206)  Sandmeyer:  Ueber  die  Folgen  der  partiellen  Pankreasextirpation 
beim  Hund.     Zeitschrift  fiir  Biologie,  V.  31,  p.  32,  (1895). 

(207)  Sawamura:  tjber  Enzyme  im  Verdauungskanal.  Biilletin  of  the  Col- 
lege of  Agriculture,  Tokyo  Imperial  University,  V.  No.  2,  (1902);  Jahresbericht 
fur  Thierchemie,  V.  32,  p.  419,  (1902). 

(208)  Schellenberg:  Untersuchungen  iiber  das  Verhalten  einiger  Pilze  gegen 
Hemicellulose.  Flora,  V.  98,  p.  257,  (1908).  Biochemisches  Centralblatt,  V.  7, 
p.  767,   (1907). 

(209)  Schmidt,  A.:  Neue  Beobachtungen  zur  Erklarung  und  rationellen 
Behandlung  der  chronischen  habituellen  Obstipation.  Miinchener  medizinische 
Wochenschrift,  V.  52,  p.  1970,  (1905). 

(210)  Schmidt,  C.  :  Ueber  Pflanzenschleim  und  Bassorin.  Liebig's  Annalen, 
V.  51,  p.  56,  (1844). 

(211)  Schmidt  .and  Lohrisch  :  Weitere  Beobachtungen  iiber  die  Bedeutung  der 
Zellulose  (Hemicellulose)  fur  die  Ernahrung  der  Diabetiker.  Deutsche  medizi- 
ische  Wochenschrift,  No.  47,  p.  1,  (1908). 

(212)  Schuize:  Ueber  die  Zellwandbestandtheile  der  Cotyledonen  von  Lupi- 
nus  luteus  und  Lupinus  angustifoHus,  und  uber  ihr  Verhalten  wahrend  des  Keim- 
ungsvorganges.     Zeitschrift  fiir  physiologische  Chemie,   V.   21,  p.  392,    (1896). 

(213)  Schulze  :  Uber  die  zur  Gruppe  der  stickstofffreien  Extractstoffe  gehoren- 
den  Pflanzenbestandtheile.     Journal  fur  Landmrtschaft,  V.  52,  (1904). 

(214)  Schulze:  Zur  Kenntnis  des  /8-Galactans.  Berichte  der  deutschen 
chemischen  GeseUschaft,  V.  25,  p.  2213,  (1893). 

(215)  Schulze:  Ueber  das  Vorkommen  eines  unloshchen  Schleimsaure 
gebenden  Kohlehydrats  in  den  Rothklee-  und  Luzeme-pflanzen.  Die  Landwirt- 
schaftHche  Versuchs-Stationen,  V.  42,  p.  9,  (1893). 

(216)  Schuxze:  Zur  Kenntnis  der  in  den  Leguminosensamen  enthaltenen 
Kohlehydrate.     Ibid.,  V.  41,  p.  207,  (1892). 

(217)  Schulze,  Steiger  and  Maxwell:  Zur  Chemie  der  Pflanzenzellmem- 
branen.     Zeitschrift  fiir  physiologische  Chemie,  V.  14,  p.  227,  (1890). 


376  Mary  Davies  Swartz, 

(218)  SCHULZE  AND  Castoro:  Beitrage  zur  Kenntnis  der  Hemicellulosen. 
Ibid.,  V.  37,  p.  41,  (1902);  V.  39,  p.  318,  (1904). 

(219)  ScHULZE  .AND  Steiger:  Untersuchungen  liber  die  stickstofffreien  Reser- 
vestoffe  der  Samen  von  Lupinus  luteus  und  iiber  die  Umwandlungen  derselben 
wahrend  des  Keimungsprozesses.  Die  Landwirtschaftliche  Versuchs-Stationen, 
V.  36,  p.  391,  (1889). 

(220)  Sebor:  Ueber  die  Kohlenhydrate  des  Carragheen-Moos.  Botanisches 
Centralblatt,  V.  86,  p.  70,  (1901). 

(221)  Strauss:  Ueber  das  Vorkommen  eini  gar  Kohlehydratefermente  bei 
Lepidopteren  und  Dipteren  in  verschiedenen  Entwicklungsstadien.  Zeitschrift  fiir 
Biologic,  V.  52,  p.  95,  (1908). 

(222)  Ul.ander:  Untersuchungen  iiber  die  Kohlenhydrate  der  Flechten. 
Dissertation,  Gottingen,   1905. 

(223)  Voit:  Ueber  die  Glykogenbildung  nach  Aufnahme  verschiedener 
Zuckerarten.     Zeitschrift  fiir  Biologie,  V.  28,  p.  245,   (1891). 

(224)  Voit:  Ueber  das  Verhalten  der  Galactose  beim  Diabetiker.  Zeit- 
schrift fiir  Biologie,  V.  29,  p.   147,  (1892). 

(225)  Von  Planta  and  Schulze:  Ueber  ein  neues  krystaUisirbares  Kohle- 
hydrat.     Berichte  der  deutschen  chemischen  Gesellschaft,  V.  23,  p.  1692,  (1890). 

(226)  Weiniand:  Ueber  die  Bildung  von  Glykogen  nach  Galactose-fiitterung. 
Zeitschrift  fur  Biologie,  V.  40,  p.  374,  (1900). 

V.  mannans. 

(227)  Bertrand:  Sur  la  presence  de  la  mannocellulose  dans  le  tissu  ligneux 
des  plantes  gymnospermes.     Comptes  Rendus,  V.  129,  p.  1025,  (1899). 

(228)  BiERRY  and  Gi.aja:  Sur  la  digestion  des  mannanes  et  des  galactanes. 
Comptes  Rendus  de  la  Societe  de  Biologie,  V.  60,  p.  945,  (1906). 

(229)  BiERRY  AND  GiAjA:  Digestion  des  mannanes  et  des  galactanes. 
Comptes  Rendus,  V.  148,  pp.  735  and  507,  (1909). 

(230)  Brown  and  Morris:  On  the  Existence  of  a  Cellulose-dissolving  Enzyme 
(Cytohydrolyst)  in  the  Germinating  Seeds  of  the  Grasses.  Journal  of  the  Chemical 
Society,  London,  V.  57,  p.  497,  (1890). 

(231)  Bourquelot  and  Herissey:  Sur  la  composition  de  I'albumen  de  la 
graine  du  Phoenix  canariensis  et  sur  les  phenomenes  chimiques  qui  accompagnent 
la  germination  de  cette  graine.     Comptes  Rendus,  V.  133,  p.  302,  (1901). 

(232)  Bourquelot  and  Herissey:  Sur  la  composition  de  I'albimien  de  la 
graine  de  caroubier:  production  de  galactose  et  de  mannose  par  hydrolyse.  Ibid., 
V.  129,  p.  228,  (1899).  Sur  la  composition  de  la  graine  de  caroubier.  Ibid.,  V.  129, 
p.  391,   (1899). 

(233)  Bourquelot  and  Herissey:  Germination  de  la  graine  de  caroubier: 
production  de  mannose  par  un  ferment  soluble.     Ibid.,  V.  129,  p.  614,     (1899). 

(234)  Bourquelot  and  Herissey:  Sur  les  ferments  solubles  produits  pendant 
la  germination  par  les  graines  a  albumen  corne.  Comptes  Rendus,  V.  130,  p.  42, 
(1900). 

(235)  Bourquelot  and  Herissey:  Sur  I'individuahte  de  laseminase,  ferment 
soluble  secrete  par  les  graines  de  legumineuses  a  albumen  corne  pendant  la  germina- 
tion.   Ibid.,  V.  130,  p.  340,  (1900). 


Nutrition  Investigations.  Zll 

(236)  BoxiRQUELOT  AND  Herissey:  Sur  la  mecanisme  de  la  saccharification 
des  mannanes  du  corrozo  par  la  seminase  de  la  Luzerne.  Ibid.,  V.  136,  p.  404, 
(1903). 

(237)  Castoro:  Beitrage  zur  Kenntnis  der  Hemicellulosen.  Zeitschrift  ftir 
physiologische  Chemie,  V.  49,  p.  96,  (1906). 

(238)  Crjemer:  Verhalten  einiger  Zuckerarten  im  tierischen  Organismus. 
Zeitschrift  fur  Biologic,  V.  29,  p.  484,  (1892). 

(239)  Dillingham:  A  Contribution  to  the  History  of  the  Use  of  Bark  Bread. 
Bulletin  of  the  Bussey  Institution,  Vol.  Ill,  Part  V.  120,  (1906). 

(240)  Dubat:  Composition  des  hydrates  de  carbone  de  reserve  de  I'albumen 
des  graines  de  quelques  Liliacees  et  en  particiilier  du  Petit  Haux.  Comptes  Rendus, 
V.  133,  p.  942  (1901). 

(241)  Effront:  Sur  une  nouvelle  enzj^me  hydrolytique,  "la  caroubinase." 
Ibid.,  V.  125,  pp.  38  and  116,  (1897).  Sur  la  caroubinase.  Ibid.,  V.  125,  p.  309, 
(1897). 

(242)  Fischer  and  PIirschberger:  Ueber  Mannose.  Berichte  der  deutschen 
chemischen  Gesellschaft,  V.  21,  p.  1805,  (1888);  V.  22,  pp.  365  and  1155,  (1889). 

(243)  Franck  :  Ueber  die  anatomische  Bedeutung  und  die  Enstehung  der 
vegetabilischen  Schleime.  Jahrbiicher  fiir  wissenschaftliche  Botanik,  V.  5,  p.  161, 
(1866). 

(244)  Gans  and  Tollens:  Ueber  die  Bildung  von  Zuckersaure  aus  Dextrose 
haltenden  Stoffen,  besonders  aus  Raffinose,  und  iiber  die  Untersuchung  einiger 
Pflanzenschleimarten.     Liebig's  Annalen,  V.  249,  p.  215,  (1888). 

(245)  Gatin:  x\ction  de  quelques  diastases  animales  sur  certaines  mannanes. 
Comptes  Rendus  de  la  Societe  de  Biologic,  V.  58,  p.  847,  (1905). 

(246)  Gatin  and  Gatin:  tJber  die  Verdaulichkeit  der  Mannanen  durch  Dias- 
tasen  der  hoheren  Tiere.     Chemisches  Centralblatt,   1907  (2),  p.  1181. 

(247)  Gatin:  Isomerisation  de  mannose  en  glycose  sous  Taction  d'un  fermeni 
soluble.     Comptes  Rendus  de  la  Societe  de  Biologic,  V.  64,  p.  903,  (1908). 

(248)  Girand:  Etude  comparative  des  gommes  et  des  mucilages.  Comptes 
Rendus,  V.  80,  p.  477,  (1875). 

(249)  Goeet:  Sur  la  composition  de  I'albumen  de  la  graine  de  fevicr  d'Ameri- 
que  (Gleditschia  triacanthos  L.,  Legumineuses) .  Comptes  Rendus,  V.  131,  p.  60, 
(1900). 

(250)  Gruss:  Studien  iiber  Reserve-Cellulose.  Botanisches  Centralblatt, 
V.  70,  p.  242,  (1897). 

(251)  Gruss:  Ueber  den  Umsatz  bei  der  Keimung  der  Dattel.  Berichte  der 
deutschen  botanischen  Gesellschaft,  V.  20,  p.  36,  (1902). 

(252)  Herissey:  Sur  la  digestion  de  la  mannane  des  tubercules  d'Orchidees. 
Comptes  Rendus,  V.  134,  p.  721,  (1902). 

(253)  Herissey:  Recherches  chimiques  et  physiologiques  sur  la  digestion  des 
mannanes  et  galactanes  par  la  seminase  chez  les  vegetaux.  Revue  Generale  de 
Botanique,  1903,  p.  345. 

(254)  Hilger:  Zur  Kenntnis  der  Pfianzenschleime.  Berichte  der  deutschen 
chemischen  Gesellschaft,  V.  36,  p.  3198,  (1903). 

(255)  Kano  and  Iishima:  Bulletin  of  the  College  of  Agriculture.  Tokyo  Im- 
perial University,  II,  No.  2,  1894.  (Cited  by  Day,  Bulletin  202.  Office  of  Ex- 
periment Stations,  United  States  Department  of  Agriculture.) 


378  Mary  Davies  Swartz, 

(256)  Kinoshita:  On  the  Occurrence  of  two  kinds  of  Mannan  in  the  Root  of 
Conophallus  Konjaku.  Bulletin  of  the  College  of  Agriculture,  Tokyo  Imperial 
University,  No.  V,  p.  205,  (1902). 

(257)  Kinoshita:  On  the  Occurrence  of  Mannan.  Ibid.,  No.  V,  p.  253, 
(1902). 

(258)  Meigen  and  Spreng:  Ueber  die  Kohlehydrate  der  Hefe.  Zeitschrift 
fiir  phj'siologische  Chemie,  V.  55,  p.  48,  (1908). 

(259)  Mulder:  Ueber  Pflanzenschleim.  Journal  fiir  praktische  Chemie,  V. 
37,  p.  334,  (1846). 

(260)  Nexjberg  and  Mayer:  Schicksal  der  drei  Marmosen  im  Kaninchenleib. 
Zeitschrift  fiir  physiologische  Chemie,  V.  37,  p.  530,  (1902). 

(261)  Newcombe:  Cellulose  Enzymes.    Annals  of  Botany,  V.  13,  p.  49,  (1899). 

(262)  Niebling:  Untersuchungen  iiber  die  kiinstliche  Verdauung  landwirth- 
schafthcher  Futtermittel  nach  Stutzer,  imd  Pepsinwarkungen  im  allgemeinen. 
Landwirtschaftliche  Jahrbucher,  V.  19,  p.  149,   (1890). 

(263)  Pohl:  Ueber  die  FaUbarkeit  coUoider  Kohlenhydrate  durch  Salze. 
Zeitschrift  fur  phj'siologische  Chemie,  V.  14,  p.  159,  (1890). 

(264)  Reiss:  Ueber  die  Natur  der  Reserve-Cellulose.  Berichte  der  deutschen 
botanischen  Gesellschaft,  V.  7,  p.  322,  (1889). 

(265)  Roseneeld:  Untersuchungen  iiber  Kolilehydrate.  Centralblatt  fiir 
innere  Medizin,  V.  21,  p.  177,  (1900). 

(266)  Sachs:  Zur  Keimungsgeschichte  der  Dattel.  Botanische  Zeitung, 
1862,  p.  241. 

(267)  Sawamxtra:  On  the  Liquefaction  of  Mannan  by  Microbes.  Central- 
blatt fur  Bakteriologie,  Abtheilung  II.,  V.  11,  p.  21,  (1903-4). 

(268)  Sawamura:  On  the  Digestive  Power  of  the  Intestinal  Canal.  Bulletin 
of  the  College  of  Agricultmre,  Tokyo  Imperial  University,  No.  V,  p.  155,    (1902). 

(269)  Schellenberg  :  Untersuchungen  iiber  das  Verhalten  einiger  Pilze 
gegen  Hemicellulosen.     Flora,  V.  98,  p.  257,  (1908). 

(270)  Schmidt,  C:  Ueber  Pflanzenschleim  und  Bassorin.  Liebig's  Annalen 
V.  51,  p.  29,  (1844). 

(271)  Storer:  Testing  for  Mannose.  Bulletin  of  the  Bussey  Institution, 
Vol.  Ill,  Part  II,  p.  13,  (1902). 

(272)  Storer:  Notes  on  the  Occurrence  of  Mannan  in  the  Wood  of  Some  Kinds 
of  Trees,  and  in  Various  Roots  and  Fruits.     Ibid.,  V.  Ill,  Part  III,  p.  47,  (1903). 

(273)  Schulze:  Zur  Chemie  der  pflanzlichen  Zellmembranen.  Zeitschrift  fiir 
physiologische  Chemie,  V.  16,  p.  387,  (1892). 

(274)  Schuster  and  Liebscher:  Der  Nahrwerth  der  Steinnussspahne. 
Landwirthschaftliche  Jahrbiicher,  V.  19,  p.  143,  (1890). 

(275)  STR.A.USS:  Ueber  das  Vorkommen  einiger  Kohlehydratefermente  bei 
Lepidopteren  und  Dipteren  in  verschiedenen  Entwicklungsstadien.  Zeitschrift  fiir 
Biologie,  V.  52,  p.  95,  (1908). 

(276)  Thamm:  Ein  Beitrag  zur  Kenntnis  der  Pflanzenschleime.  Dissertation, 
Munchen,  1903. 

(277)  ToLLENS  AND  Gans:  Mannose  oder  Isomanitose  aus  Salepschleim. 
Berichte  der  deutschen  chemischen  Gesellschaft,  V.  21,  p.2150,   (1888). 

(278)  ToLLENS  AND  OsHiMA:  Ubcr  das  Nori  aus  Japan.  Berichte  der  deut- 
schen chemischen  Gesellschaft,  V.  34,  p.  1422,  (1901). 

(279)  ToLLENS  AND  WiDSTOE :  Ubcr  die  Reactionen  des  Methvl-Furfurols  und 
der  Methyl-Pentosane.     Ibid.,  V.  33,  p.  132,  (1900). 


Nutrition  Investigations.  379 

(280)  Tsuji:  Mannan  as  an  Article  of  Human  Food.  Bulletin  of  the  College 
of  Agriculture,  Tokyo  Imperial  University,  No.  II,  p.  103,  (1894). 

(281)  TsuKAiiATo:  Ueber  die  Bildung  von  Mannan  in  Amorphophallus 
Konjak.     Chemisches  Centralblatt,  V.  97a,  p.  930,  (1897). 

(282)  Van  Ekenstein:  Sur  la  caroubinose  et  sur  la  d-mannose.  Comptes 
Rendus,  V.  125,  p.  719,  (1897). 

(283)  Voit:  Ueber  die  Aufnahme  des  Pflanzenschleims  und  des  Gummis  aus 
dem  Darme  in  die  Safte.     Zeitschrift  fur  Biologie,  V.  10,  p.  59,  (1874). 

(284)  Weiske:  Versuche  iiber  die  VerdauHchkeit  und  den  Nahreffect  des 
Jobannisbrodes.     Journal  fiir  Landwirthschaft,  V.  27,  p.  321,  (1879). 

(285)  Zanotti:  Untersuchungen  iiber  einige  zusammengesetzte  KoDaehy- 
drate.     Chemisches  Centralblatt,  V.  99a,  p.  1209,  (1899). 

VI.      LEVULANS. 

(286)  Bierry:  Recherches  sur  la  digestion  de  I'inuline.  Comptes  Rendus  de 
la  Societe  de  Biologie,  V.  59,  p.  256,  (1905). 

(287)  Blerry:  Recherches  sur  la  digestion  de  I'iniiline.  Comptes  Rendus, 
V.  150,  p.   116,   (1910). 

(288)  BiERRi  AND  Porter:  Recherches  sur  la  digestion  de  I'inuline.  Ibid., 
V.  52,  p.  423,  (1900). 

(289)  Bourquelot:  Inulase  et  fermentation  alcoholique  indirecte  de  I'inuline. 
Comptes  Rendus,  V.  116,  p.  1143,  (1893). 

(290)  Bourquelot:  Remarques  sur  les  ferments  solubles  secretes  par  1' As- 
pergillus et  le  Penicilliima.  Comptes  Rendus  de  las  Societe  de  Biologie,  V.  9, 
p.  653,  (1893). 

(291)  Chevastelon:  Sur  I'inuline  d'ail,  de  la  jacinthe,  de  I'asphodele  et  de  la 
tubereuse.     Journal  de  Pharmacie,  V.  4,  p.  2,  (1895).     (Cited  by  Dean.) 

(292)  Chittenden:  The  Behavior  of  Inuhn  in  the  Gastro-intestinal  Tract. 
American  Jommal  of  Physiology,   V.  2,  p.  XVII,  (1898). 

(293)  Dean:  Experimental  Studies  on  Inulase.  Botanical  Gazette,  V.  35, 
p.  24,  (1903). 

(294)  Dean:  On  Inulin.     American  Chemical  Journal,  V.  32,  p.   69,  (1904). 

(295)  Due  amp:  Beitrag  ztmi  Studium  der  Unterscheidung  des  Colibazillus, 
Wirkung  der  BaziUen  der  CoHtyphusruhrgruppe  auf  die  Kohlehydrate.  Jahres- 
bericht  fur  Thierchemie,  V.  37,  p.  952,  (1907). 

(296)  Eestrand  AND  Johanson:  ZurKenntnis  der  Kohlehydrate,  I.  Berichte 
der  deutschen  chemischen  Gessellschaft,  V.  20,  p.  3310,  (1887).  Zur  Kenntnis  der 
Kohlehydrate,  II.    Ibid.,  V.  21,  p.  594,  (1888). 

(297)  Finn:  ExperimenteHe  Beitrage  zur  Glycogen-  und  Zuckerbildimg  in  der 
Leber.  Arbeiten  aus  dem  physiologischen  Laboratorium,  Wiirzburg,  1877.  (Cited 
by  Miura.) 

(298)  FiTz:  Ueber  Schizomycetengahrungen.  Berichte  der  deutschen  chemi- 
schen GeseUschaft,  V.  11,  p.  42,  (1878). 

(299)  Frerichs:  Zur  Glykogenbildung  in  der  Leber.  Dissertation,  Wiirz- 
burg, 1876.     (Cited  by  Miura.) 

(300)  Green:  On  the  Germination  of  the  Jerusalem  Artichoke  (Helianth  us 
tuberosus).     Annals  of  Botany,  V.  I,  p.  223,  (1888). 

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380  Mary  Davies  Swartz, 

(302)  KiLiANi:    Ueber  Inulin.     Liebig's  Annalen,  V.  205,  p.   145,   (1880). 

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chemie,    V.6,  p.  180,  (1876). 

(304)  Robert:  Ueber  einige  Enzyme  wirbelloser  Tiere.  Pfliiger's  Archiv  fiir 
Physiologie,  V.  99,  p.  116,  (1903). 

(305)  KiJLz:  Beitrage  zur  Pathologie  und  Therapie  des  Diabetes  Mellitus. 
Jahresbericht  fur  Thierchemie,  V.  4,  p.  448,  (1874). 

(306)  KuLZ:  Beitrage  zur  Kenntnis  des  Glycogens.  Centralblatt  fur  Physio- 
logie,  1890,  p.  789. 

(307)  lAvY:  De  la  fermentation  alcoholique  des  topinambours,  sous  I'influence 
des  levures.     Comptes  Rendus,  V.  116,  p.  1381,  (1893). 

(308)  Lindner:  Gar\'ersuche  mit  verschiedenen  Hefen  und  Zuckerarten. 
Chemisches  Centralblatt,  1901,  p.  56. 

(309)  V.  Lippmann:  Ueber  das  Lavulan,  eine  neue,  in  der  Melasse  der  Riiben- 
zucker-fabriken  vorkommende  Gummiart.  Berichte  der  deutschen  chemischen 
GeseUschaft,  V.  11,  p.  57.  (1881). 

(310)  Luchsinger:  Zur  Glykogenbildung  in  der  Leber.  Pfliiger's  Archiv  fiir 
Physiologie,  V.  8,  p.  289,  (1874). 

(311)  Mendel  and  Mitchell:  On  the  Utihzation  of  Various  Carbohydrates 
without  Intervention  of  the  i\limentary  Digestive  Processes.  American  Journal 
of  Physiology,  V.  14,  p.  239,  (1905). 

(312)  Mendel  and  Nakaseko:  Glycogen  Formation  after  Inuhn  Feeding. 
Ibid.,  V.  4,  p.  246,  (1900). 

(313)  Miura:  Wird  durch  Zufuhr  von  Inulin  die  Glykogenbildung  gesteigert? 
Zeitschrift  fur  Biologic,  V.  32,  p.  255,  (1895). 

(314)  Reidemeister:  Sinistrin,  Levulin,  und  Triticin.  Chemisches  Central- 
blatt, 1880,  p.  808;  Jahresbericht  fur  Thierchemie,  V.  11,  p.  68,  (1881). 

(315)  Richaud:  Sur  quelques  points  relatifs  a  I'histoire  physiologique  de 
I'inuline  chez  les  animaux.  Comptes  Rendus  de  la  Societe  de  Biologic,  V.  52,  p. 
416,  (1900). 

(316)  Saiki:  Anti-inulase.  Journal  of  Biological  Chemistry,  V.  3,  p.  395, 
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(317)  Sandmeyer:  Ueber  die  Folgen  der  partiellen  Pancreasextirpation  beim 
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(318)  Schmiedeberg  :  Ueber  ein  neues  Kohlehydrat.  Zeitschrift  fiir  physio- 
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(319)  Strauss:  Ueber  das  Vorkommen  einiger  Kohlehydrate  fermente  bei 
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(320)  Tanret:  Sur  la  levulosane,  nouveau  principe  immediat  des  cereales 
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(321)  Tanret:  Sur  I'inuline  et  deux  principes  immediats  nouveaux.  Ibid., 
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(322)  Tanret:  Sur  les  hydrates  de  carbone  du  topinambour.  Ibid.,  V.  117, 
p.  50,  (1893). 

(323)  Tanret:  Sur  une  nouvelle  glucosane,  la  levoglucosane.  Ibid.,  V.  119, 
p.  158,  (1894). 

(324)  Von  Mering:  Zur  Glykogenbildung  in  der  Leber.  Pfliiger's  Archiv 
fiir  Physiologie  V.  14,  p.  274,  (1877). 


Nutrition  Investigations.  381 

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(327)  Went:  Monilia  sitophila  (Mont.)  Sacc,  ein  technischer  Pilz.  Che- 
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vn.    dextrans. 

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(330)  Bauer:  Ueber  eine  aus  Laminariaschleim  entstehende  Zuckerart. 
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(331)  Bauer:  Ueber  die  Arabonsaure  und  die  aus  Lichenin  entstehende 
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(332)  Berg:  Jahresbericht  fiir  Chemie,  1873,  p.  848.  (From  Russ.  Zeitschr. 
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(333)  Berzelius:  Recherches  sur  la  nature  du  lichen  d'Islande,  et  sur  son 
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(334)  Brown:  Notes  on  Cetraria  Islandica.  American  Journal  of  Physiology, 
V.  I,  p.  455,  (1898). 

(335)  Escombe:  Chemie  der  Membranen  der  Flechten  und  Pilze.  Zeitschrift 
fiir  physiologische  Chemie,  V.  22,  p.  288,   (1896). 

(336)  HoNiG  AND  St.  Schubert:  Ueber  Lichenin.  Monatshefte  fiir  Chemie, 
V.  8,  p.  452,  (1887). 

(337)  Klason:  tjber  die  durch  Inversion  von  Lichenin  entstehende  Zucker. 
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(338)  Meyer:  Ueber  ReservestoCfe,  Kerne  und  Sporenbildung  der  Bakterien. 
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(340)  Mendel:  Das  Verhalten  einiger  unverdaulicher  Kohlehydrate  im  Ver- 
dauungstrakt.  Zentralblatt  fiir  die  gesammte  Physiologie  und  Pathologic  des 
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(341)  Muller,  Karl:  Die  Chemische  Zusammensetzung  der  Zellmembranen 
bei  vcrschiedenen  Kryptogamen.  Zeitschrift  fiir  physiologische  Chemise,  V. 
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(342)  Nilson:  Kenntnis  der  Kohlcnhydrate  in  den  Flechten.  Upsala 
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53,  (1893). 

(343)  O'Sullivan:  Amylam  in  Wheat,  Rye  and  Barley.  Chemical  News, 
V.  44,  p.  258,  (1881). 

(344)  Poulsson:  Untersuchungen  iiber  das  Verhalten  einiger  Flechten- 
kohlehydrate  im  menschlichen  Organismus  und  iiber  die  Anwendung  derselben 
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382 


Mary  Davies  Swarlz. 


(345)  RoTHENFUSSER :  Der  Schleimkorper  des  Leinsamens.  Dissertation, 
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(347)  Tortjp:  A  New  Carbohydrate  from  Laminaria  Digitata.  Biochemisches 
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(348)  Ulander:  Untersuchungen  iiber  die  Kohlenhydrate  der  Flechten. 
Dissertation,   Gottingen,   (1905). 

(349)  Voit:  Hermann's  Handbuch  der  Physiologie,  V.  6,  p.  413,   (1881). 

(350)  Winterstein:  Zur  Kenntnis  der  in  den  Membranen  der  Pilze  enthal- 
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(351)  Von  Mering:  Zur  Glykogenbildimg  in  der  Leber.  Pfliiger's  Archiv 
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(352)  Yoshimura:  Ueber  einige  Pflanzenschleime.  Jahresbericht  fiir  Thier- 
chemie, V.  25,  p.  51,  (1895).  (Bulletin  II,  No.  4,  College  of  Agriculture,  Tokyo 
Imperial    University.) 


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